Methods for detection of biological substances

ABSTRACT

Methods and compositions are provided for detection of biological substances in nasal specimen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/618,882, filed Sep. 14, 2012, now U.S. Pat. No. 8,663,938, whichis a continuation application of U.S. application Ser. No. 12/523,040,now U.S. Pat. No. 8,293,489, which was the National Stage ofInternational Application No. PCT/US08/52712, filed Jan. 31, 2008, whichin turn claims benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication No. 60/915,008, filed Apr. 30, 2007 and U.S. ProvisionalApplication No. 60/887,587 filed Jan. 31, 2007, each of which isincorporated by reference herein for all purposes.

BACKGROUND OF THE INVENTION

Detection and identification of biological substances in tissue samplesis used for the diagnosis, prognosis, and monitoring of diseases.Efficient identification of biological substances aids in devisingeffective treatment strategies.

Most of the current diagnostic techniques involve invasive proceduresfor the removal of tissue samples or blood. Hence, there is need for thedevelopment of minimally invasive procedures for biological sampleretrieval.

The present invention provides methods and compositions for detection ofbiological substances, diagnosis of diseases based on this detection andmethods for treatment of the diseases after the diagnosis.

SUMMARY OF THE INVENTION

In one embodiment, a method is provided for detecting a biologicalsubstance or element from a nasal specimen of a patient to detect abiological substance or element that is associated with a disease orcondition, and where the disease is not a respiratory disease.

In one aspect, the detected biological substance is agouti relatedprotein, alpha fetoprotein (AFP), brain derived neurotrophic factor(BDNF), bone morphogenetic protein-2 (BMP-2), ciliary neurotrophicfactor (CNTF), thymus and activation-regulated chemokines (CCL17/TARC)CC chemokines, cystatin, D-dimer, E selectin, endoglin, epidermal growthfactor, (EGF), endothelial nitric oxide synthase, (eNOS), FAS ligand,fibroblastic growth factor basic (FGF basis), granulocyte macrophagecolony stimulating factor (GM-CSF), hepatocyte growth factor (HGF),inducible nitric oxide synthase (iNOS), insulin-like growth factor 1(IGF-1), interferon alpha (INF-α), interferon beta (INF-β), interferongamma (INF-γ), interferon omega (INF-ω), intracellular adhesion molecule1 (ICAM-1), interleukin-1 (IL-1), interleukin-1 receptor (IL-1receptor), interleukin-2 (IL-2), interleukin-2 receptor (IL-2 receptor),interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-15 (IL-15),interleukin-17 (IL-17), interleukin-18 (IL-18), keratinocyte growthfactor (KGF), L-selectin, leptin, leukemia inhibitor factor (LIF),matrix metalloproteinase 1 (MMP-1), migrating inhibitory factor (MIF),nerve growth factor (NGF), P selectin, placental growth factor (PlGF),platelet derived growth factor-AA (PDGF-AA), platelet derived growthfactor-BB (PDGF-BB), pro-B type natiuretic peptide, receptor forabdominal glycation end product (RAGE), stem cell factor (SCF),substance P, triggering receptor expressed on myeloid cells (TREM-1),transforming growth factor alpha (TGF-alpha), transforming growth factorbeta (TGF-beta), tumor necrosis factor (TNF), tumor necrosis factorreceptor 1 (TNF-R1), tumor necrosis factor receptor 2 (TNF-R2),TNF-related apoptosis-inducing ligand (TRAIL), vascular cell adhesionmolecule 1 (VCAM1), vascular endothelial growth factor C (VEGF-C),vascular growth factor D (VEGF-D), vascular endothelia growth factorreceptor 1 (VEGFR1), or vascular endothelia growth factor receptor 2(VEGFR2).

In another aspect, the detected biological substance is a nucleic acid,a protein, a carbohydrate, a lipid, a metabolite, a hormone, or acombination of these. In a further aspect, the biological substance is aprotein. In another aspect, the protein is detected by immunoassay, massspectrometry, liquid chromatography, electrophoresis, arrays, orbiologic sensors. In one aspect detection of a biological substance orelement is performed on a point of care device.

In one aspect, the detected element is a metal. In another element themetal is copper or zinc.

In one aspect, the disease or condition associated with a detectedbiological substance or element is preeclampsia, a bacterial infection,a viral infection, a parasitic infection, a metabolic disease, agastrointestinal disease, a cardiovascular disease, a neurologicdisease, a hematologic disease, an endocrine disease, a malignantdisease, an autoimmune disease, or an inflammatory disease.

In another aspect, the method also includes diagnosing a disease orcondition. In a still further aspect, the method includes administeringa therapeutically effective amount of a drug or active agent to treatthe diagnosed disease or condition. In another aspect, theadministration route is oral administration, transmucosaladministration, buccal administration, nasal administration, parentaladministration, intravenous administration, subcutaneous administration,intramuscular administration, transdermal administration, or rectaladministration. In one aspect, the administration route is by nasaladministration.

In one embodiment, a method is provided to detect a biological substanceor element in nasal mucus that is associated with the likelihood ofoccurrence of a disease or condition, and where the disease is not arespiratory disease.

In one aspect, the biological substance associated with the likelihoodof occurrence of a disease is agouti related protein, alpha fetoprotein(AFP), brain derived neurotrophic factor (BDNF), bone morphogeneticprotein-2 (BMP-2), ciliary neurotrophic factor (CNTF), thymus andactivation-regulated chemokines (CCL17/TARC) CC chemokines, cystatin,d-dimer, E selectin, endoglin, epidermal growth factor, (EGF),endothelial nitric oxide synthase, (eNOS), FAS ligand, fibroblasticgrowth factor basic (FGF basis), granulocyte macrophage colonystimulating factor (GM-CSF), hepatocyte growth factor (HGF), induciblenitric oxide synthase (iNOS), insulin-like growth factor 1 (IGF-1),interferon alpha (INF-α), interferon beta (INF-β), interferon gamma(INF-γ), interferon omega (INF-ω), intracellular adhesion molecule 1(ICAM-1), interleukin-1 (IL-1), interleukin-1 receptor (IL-1 receptor),interleukin-2 (IL-2), interleukin-2 receptor (IL-2 receptor),interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-15 (IL-15),interleukin-17 (IL-17), interleukin-18 (IL-18), keratinocyte growthfactor (KGF), L-selectin, leptin, leukemia inhibitor factor (LIF),matrix metalloproteinase 1 (MMP-1), migrating inhibitory factor (MIF),nerve growth factor (NGF), P selectin, placental growth factor (PlGF),platelet derived growth factor-AA (PDGF-AA), platelet derived growthfactor-BB (PDGF-BB), pro-B type natiuretic peptide, receptor forabdominal glycation end product (RAGE), stem cell factor (SCF),substance P, triggering receptor expressed on myeloid cells (TREM-1),transforming growth factor alpha (TGF-alpha), transforming growth factorbeta (TGF-beta), tumor necrosis factor (TNF), tumor necrosis factorreceptor 1 (TNF-R1), tumor necrosis factor receptor 2 (TNF-R2),TNF-related apoptosis-inducing ligand (TRAIL), vascular cell adhesionmolecule 1 (VCAM1), vascular endothelial growth factor C (VEGF-C),vascular growth factor D (VEGF-D), vascular endothelia growth factorreceptor 1 (VEGFR1), or vascular endothelia growth factor receptor 2(VEGFR2).

In another aspect, the biological substance associated with thelikelihood of occurrence of a disease is a nucleic acid, a protein, acarbohydrate, a lipid, a metabolite, a hormone, or combination of theabove. In another aspect, the biological substance is a protein. In afurther aspect, the protein is detected by immunoassay, massspectrometry, liquid chromatography, electrophoresis, arrays, orbiologic sensors. In one aspect, the biological substance or element isdetected using a point of care device. In a further aspect, the point ofcare diagnostic device is a lateral-flow immunoassay.

In one aspect, the element associated with the likelihood of occurrenceof a disease is a metal. In a further aspect, the metal is copper orzinc.

In one aspect, the disease or condition associated with the detectedbiological substance or element is preeclampsia, a bacterial infection,a viral infection, a parasitic infection, a metabolic disease, agastrointestinal disease, a cardiovascular disease, a neurologicdisease, a hematologic disease, an endocrine disease, a malignantdisease, an autoimmune disease, or an inflammatory disease. In anotheraspect, a prediction is made of the likelihood that the disease orcondition will occur.

In one aspect, the method also includes administering a therapeuticallyeffective amount of a drug or active agent to treat a predicted diseaseor condition. In another aspect, the drug or active agent isadministered by way of oral administration, transmucosal administration,buccal administration, nasal administration, parental administration,intravenous administration, subcutaneous administration, intramuscularadministration, transdermal administration, or rectal administration. Inanother aspect, the administration route is nasal administration.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a flow chart showing the steps of the methods of the presentinvention.

FIG. 2 illustrates a computer for implementing selected operationsassociated with the methods of the present invention.

FIG. 3 depicts a kit for a detection of biological substance in a nasalspecimen.

FIG. 4 depicts a polyacrylamide gel electrophoresis of samples as shownin Table 1.

FIG. 5 depict LightCycler melting peak report on results of PCR analysisof two samples of nasal mucus. Fluorescence is plotted on ordinate,temperature on abscissa.

FIG. 6 depicts LightCycler data analysis report on results of PCRanalysis of two samples of nasal mucus. Fluorescence is plotted onordinate, cycle number on abscissa.

FIG. 7 depicts LightCycler melting peak report on results of PCRanalysis of two samples of nasal mucus. Fluorescence is plotted onordinate, temperature on abscissa.

FIG. 8 depicts LightCycler data analysis report on results of PCRanalysis of two samples of nasal mucus. Fluorescence is platted onordinate, cycle number on abscissa.

FIG. 9 depicts LightCycler data analysis report on results of PCRanalysis of two samples of nasal mucus. Fluorescence is plotted onordinate, cycle number on abscissa.

FIG. 10 depicts mass spectroscopic analysis of parotid saliva ofpatients before and after treatment with rTCMS.

FIGS. 11 and 12 reflect a feedback mechanism with effects acting fromnose to brain and from brain to nose.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “diagnosis” as used herein and its grammatical equivalents,means the testing of subjects to determine if they have a particulartrait for use in a clinical decision. Diagnosis includes testing ofsubjects at risk of developing a particular disease resulting frominfection by an infectious organism or a non infectious disease, such ascancer or a metabolic disease. Diagnosis also includes testing ofsubjects who have developed particular symptoms to determine the causeof the symptoms. Diagnosis also includes prognosis, monitoring progressof a disease, and monitoring the efficacy of therapeutic regimens. Theresult of a diagnosis can be used to classify patients into groups forperformance of clinical trials for administration of certain therapies.

The term “drug” as used herein, means any compounds of any degree ofcomplexity that perturbs a biological state, whether by known or unknownmechanisms and whether or not they are used therapeutically. Drugs thusinclude: typical small molecules of research or therapeutic interest;naturally-occurring factors, such as endocrine, paracrine, or autocrinefactors or factors interacting with cell receptors of all types;intracellular factors, such as elements of intracellular signalingpathways; factors isolated from other natural sources; pesticides;herbicides; and insecticides.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. The term also refers to syntheticallygenerated nucleic acid.

The term “pathogen” as used herein includes, viral, bacterial, fungal,prion, microbial, or other material that can be detected using theteachings of the present invention. The term “pathogen” as used hereincan be natural or synthetically generated.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds. The term also refers tosynthetically generated polypeptide, peptide or protein.

The term “treating” and its grammatical equivalents as used hereininclude achieving a therapeutic benefit and/or a prophylactic benefit.By therapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a patientat risk of developing a particular disease, or to a patient reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made.

METHODS OF THE INVENTION

The present invention includes methods of analyzing samples from thenose for the detection of biological substances or elements. In someembodiments, nasal secretion or nasal mucus is collected and analyzedfor biological substances or elements. In various embodiments, theresults of this analysis can confirm the presence (qualitatively orquantitatively) of one or more biological substances or elements. Insome embodiments, confirmation of the presence of one or more biologicalsubstances or elements allows for the diagnosis or prognosis of adisease or condition and may include the determination of suitability oftherapeutic interventions. The techniques of the present invention allowfor detection of biological substances that are typically not consideredto be present in the nasal area, but are known to be present in otherbiological fluids such as blood, serum, plasma, etc. Use of nasalspecimens provides a minimally invasive manner of obtaining biologicalsamples for analysis. In some embodiments, the methods of the inventionare used to detect substances that are not present in other biologicalfluids such as blood, serum, plasma, etc., but have been now detected inthe nasal area.

The term “biological substance” as used herein, and its grammaticalequivalents, includes cells and their extra-cellular and intra-cellularconstituents. For example, biological substances include pathogens,metabolites, DNA, RNA, lipids, proteins, carbohydrates, receptors,enzymes, hormones, growth factors, growth inhibitory factors, cells,organs, tissues, portions of cells, tissues, or organs, subcellularorganelles, chemically reactive molecules like H⁺, superoxides, ATP,citric acid, protein albumin, as well as combinations or aggregaterepresentations of these types of biological variables. In addition,biological substances can include therapeutic agents such asmethotrexate, steroids, non-steroidal anti-inflammatory drugs, solubleTNF-alpha receptor, TNF-alpha antibody, and interleukin-1 receptorantagonists.

A first aspect of the present invention is a method of detecting abiological substance in a nasal specimen, wherein the biologicalsubstance is not related to a respiratory disease (e.g., caused by apathogen). Respiratory diseases caused by pathogens include upperrespiratory tract viral infection, upper respiratory tract bacterialinfection, bacterial sinusitis, and whooping cough. However, in someembodiments, the methods of the present invention are suitable fordetection of substances related to respiratory diseases that are notcaused by pathogens, such as allergic rhinitis, asthma, and chronicobstructive pulmonary disease. Furthermore, it should be understood thatalthough some of these diseases are not caused by pathogens, they can betriggered or worsened by pathogens.

A second aspect of the invention is a method of diagnosing a disease byanalyzing a nasal specimen for a biological substance that is notrelated to a respiratory disease. In various embodiments, the results ofthis analysis can confirm presence (qualitatively or quantitatively) ofone or more biological substances. In some embodiments are then suitablefor use in diagnosis, prognosis, and determination of suitability oftherapeutic interventions.

The biological substances that can be detected by the methods of thepresent invention include, but are not limited to, insulin, insulinreceptors, ghrelin, glucose, caspases, adenylyl cyclases, carbonicanhydrases, endostatin, erythropoetin, carbonic anhydrase VI, cAMP,cGMP, nitric oxide, agouti related protein, alpha fetoprotein (AFP),brain derived neurotrophic factor (BDNF), bone morphogenetic protein-2(BMP-2), ciliary neurotrophic factor (CNTF), thymus andactivation-regulated chemokines (CCL17/TARC) CC chemokines, cystatin,d-dimer, E selectin, endoglin, epidermal growth factor, (EGF),endothelial nitric oxide synthase, (eNOS), FAS ligand, fibroblasticgrowth factor basic (FGF basis), granulocyte macrophage colonystimulating factor (GM-CSF), hepatocyte growth factor (HGF), induciblenitric oxide synthase (iNOS), insulin-like growth factor 1 (IGF-1),interferon alpha (INF-α), interferon beta (INF-β), interferon gamma(INF-γ), interferon omega (INF-ω), intracellular adhesion molecule 1(ICAM-1), interleukin-1 (IL-1), interleukin-1 receptor (IL-1 receptor),interleukin-2 (IL-2), interleukin-2 receptor (IL-2 receptor),interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-15 (IL-15),interleukin-17 (IL-17), interleukin-18 (IL-18), keratinocyte growthfactor (KGF), L-selectin, leptin, leukemia inhibitor factor (LIF),matrix metalloproteinase 1 (MMP-1), migrating inhibitory factor (MIF),nerve growth factor (NGF), P selectin, placental growth factor (PlGF),platelet derived growth factor-AA (PDGF-AA), platelet derived growthfactor-BB (PDGF-BB), pro-B type natiuretic peptide, receptor forabdominal glycation end product (RAGE), stem cell factor (SCF),substance P, triggering receptor expressed on myeloid cells (TREM-1),transforming growth factor alpha (TGF-alpha), transforming growth factorbeta (TGF-beta), tumor necrosis factor (TNF), tumor necrosis factorreceptor 1 (TNF-R1), tumor necrosis factor receptor 2 (TNF-R2),TNF-related apoptosis-inducing ligand (TRAIL), vascular cell adhesionmolecule 1 (VCAM1), vascular endothelial growth factor C (VEGF-C),vascular growth factor D (VEGF-D), vascular endothelia growth factorreceptor 1 (VEGFR1), or vascular endothelia growth factor receptor 2(VEGFR2).

In various embodiments of the invention a method is provided for thedetection of substances related to glucose metabolism such as insulinand insulin receptors. The detection of insulin, insulin receptors, andglucose is used in the diagnosis insulin resistance related conditions,such as diabetes. Use of nasal specimens for the detection of glucoseand insulin provides a minimally invasive technique for diagnosis ofdiabetes and managing diabetes care.

Another embodiment of the invention is the detection of TNFα in nasalspecimens. TNFα in nasal mucus was found to be about 30 times higherthan in saliva. The concentration of TNFα in nasal specimens, thus, canbe reflective of underlying disease processes. This detection is used inthe diagnosis of various cancers and inflammatory diseases including butnot limited to squamous cell cancer of the head and neck, breast cancer,esophageal cancer, colon cancer and liver cancer. Also, TNFα levelmonitoring in nasal specimens can be used to monitor the efficacy ofcancer and inflammatory disease therapeutics. Further, nasaladministration of anti-TNFα drugs provides a means for treatment ofdiseases in which TNFα plays a role. Also, levels of TNFα in nasalspecimens can be used to study apoptosis.

Yet another embodiment of the invention is the detection of leptin,ghrelin and agouti-related protein in nasal specimens. Also, the methodsinclude the administration of substances that modulate leptin, ghrelinand agouti-related protein for the control of appetite and treatment ofobesity and anorexia. Nasal administration of leptin can inhibitappetite and nasal administration of agouti-related protein canstimulate appetite. Antibodies to leptin, ghrelin and agouti-relatedprotein can be administered intranasally to modulate appetite. Thismodulation includes control and/or stimulation.

In some embodiments, the methods of the present invention includedetecting cAMP and cGMP in nasal specimens. Comparison of themeasurement of cAMP and cGMP in normal subjects with patients with tasteand smell loss indicated that patients with taste and smell loss haddecreased levels of cAMP in their nasal mucus. Hence, cAMP in nasalmucus can be an index of smell loss. The detection of cAMP and cGMP inthe nasal mucus provides a non-invasive method for the detection oftaste and smell loss in a subject.

A third aspect of the invention is a method of treating a disease orcondition wherein the treatment is based on the diagnosis of the diseaseby analyzing a nasal specimen for a biological substance. In oneembodiment, the disease or condition is not related to a respiratorydisease. Preferably, following the diagnosis, a therapeutic isadministered which modulates the biological specimen. In someembodiments, the treatment includes nasal administration of biologicalsubstances, such as, by way of example only, leptin, ghrelin,agouti-related protein, TNFα, insulin, or hormones. In some embodiments,the treatment includes nasal administration of therapeutic thatmodulates the identified biological substances.

In one embodiment a method is provided for treating diabetes and/orinsulin resistance, following detection of insulin, insulin receptorand/or glucose, by administering to a subject in a therapeuticallyeffective amount a drug or agent that modulates the insulin, insulinreceptor and/or glucose is administered. In one embodiment, thisadministration is via nose.

In various embodiments, a drug or agent can include antidiabetic drugs,including insulin.

In another aspect of the invention, a method is provided for treatingcancer comprising detecting one or more biological substances in a nasalspecimen, where the biological substance(s) are associated with acancer. In one embodiment for treating cancer, following detection ofTNFα, p53 or mutated p53, a drug that modulates the TNFα, p53 or mutatedp53 is administered. In one embodiment, a drug or active (e.g.,anticancer drug or agent) this administration is via nose.

In another aspect of the invention, a method is provided for treating abacterial pathogen comprising detecting one or more biologicalsubstances in a nasal specimen, where the biological substance(s) areassociated with a bacterial pathogen. In one embodiment for treatingleprosy, following detection of antibodies against mycobacterium leprae,an antibiotic drug is administered. In one embodiment, thisadministration is via nose.

In another aspect of the invention, a method is provided for treating avirus or viral infection comprising detecting one or more biologicalsubstances in a nasal specimen, where the biological substance(s) areassociated with a virus or viral infection. In one embodiment fortreating hepatitis, including hepatitis A, B, C, D, E and G, followingdetection of antibodies against hepatitis causing virus, a drug, suchas, by way of example only, interferon is administered. In oneembodiment, this administration is via nose. In one embodiment fortreating flu, following detection of flu causing pathogen or antibodiesagainst flu causing pathogen, a drug that modulates the infection isadministered. Preferably, this administration is via nose. The drug caninclude antibiotics or other drugs known in the art.

In another aspect of the invention, a method is provided for treating aweight condition or disorder comprising detecting one or more biologicalsubstances in a nasal specimen, where the biological substance(s) areassociated with a weight condition or disorder. In one embodiment amethod is provided for treating obesity, following detection of leptinor agouti-related protein, a drug that modulates the leptin oragouti-related protein is administered. The drug or agent can includeanti-agouti-related protein or leptin. In one embodiment for treatinganorexia, following detection of leptin or agouti-related protein, adrug that modulates the leptin or agouti-related protein isadministered. The drug can include anti-leptin or agouti-relatedprotein.

In another aspect of the invention, a method is provided for treating asmell loss or taste loss condition or disorder comprising detecting oneor more biological substances in a nasal specimen, where the biologicalsubstance(s) are associated with a weight condition or disorder. In oneembodiment for treating smell loss or taste loss, following detection ofTNFα, CA VI, or TRAIL, a drug that modulates the TNFα, CA VI or TRAIL isadministered. The drug or active agent can include theophylline or otherdrugs known in the art. In any of the methods of disclosed herein a drugor active agent can be administered through the nose.

The biological substances in the body interact with the brain via afeedback mechanism. The biological substances present in the nose aresecreted by glands that are controlled by a brain function and via thefeedback mechanism, these biological substances after secretion, in turnaffect the brain function. The nasal administration of the biologicalsubstance after diagnosing the disease by detecting the biologicalsubstance in the nasal secretion may be reflective of this feedbackmechanism.

In some embodiments, the treatment includes transcranial magneticstimulation (TCMS). TCMS or rTCMS (repetitive TCMS) can induce secretionof biological substances in the body, thereby inducing clinical changes.The patients suffering from loss of taste and/or smell (hypogeusiaand/or hyposmia, respectively) when treated with rTCMS, showedimprovement in their sensory acuity and decrease in their sensorydistortions. Some biological substances in these patients, such as, CAI, II and VI, zinc, and copper were found to be significantly higher inblood plasma, erythrocytes and saliva after treatment with TCMS. Theincrease of the biological substances in the body after TCMS indicatesthat TCMS induces biochemical changes in the body and can be used totreat various diseases including clinical abnormalities of sensoryfunction and neurological disorders.

The methods of the present invention disclosed herein include methodsfor detecting, diagnosing, and treating a disease in a subject (or acombination thereof), by analyzing one or more biological substances innasal tissue or secretion. The steps of the methods of the presentinvention are depicted in FIG. 1. Without limiting the scope of thepresent invention, the steps can be performed independent of each otheror one after the other. One or more steps may be skipped in the methodsof the present invention. A sample of nasal secretion is collected froma subject at step 101. One or more biological substances in the specimenis detected, measured and/or analyzed at step 102 by detectiontechniques known in the art, such as, PCR, mass spectrometry, proteinassays etc. By way of example only some of the detection techniques aredisclosed herein. A disease is diagnosed at step 103 based on thedetection, measurement and/or analysis of the biological substance. Adecision regarding treatment of the disease is made at step 104, thetreatment decision being made based on the diagnosis.

The identification of the biological substances may involve one or morecomparisons with reference specimens. The reference specimen may beobtained from the same subject or from a different subject who is eithernot affected with the disease or is a patient. The reference specimencould be obtained from one subject, multiple subjects or besynthetically generated. The identification may also involve thecomparison of the identification data with the databases to identify thebiological substance.

The steps of the methods of the present invention are provided herein.Without limiting the scope of the present invention, other techniquesfor collection of sample, detection of the biological substances anddiagnosis of the disease are known in the art and are within the scopeof the present invention.

Sample Collection

In the sample collection step, specimens from the nasal area arecollected for analysis. In some embodiments of the invention, a sampleof nasal secretions is collected directly from the nose into acollection tube or device. In other embodiments of the invention, asample of nasal secretion is collected on a sample collection device bypassing it into the nostril of a patient. The device may be insertedsequentially into each nostril of the patient and advanced parallel tothe hard palate with slow rotation. The device is then typicallytransferred to a transport tube, such as a glass or plastic test tube.The transport tube may include a suitable volume of a sterile mediumsuch as ethanol or the like.

Suitable sample collection devices are well known to those skilled inthe art. In one embodiment, a sample collection device can be a swab, awooden spatula, bibulous materials such as a cotton ball, filter, orgauze pad, an absorbent-tipped applicator, capillary tube, and apipette. Preferably, a swab can be used as a sample collection device,and the sample processing element comprises a swab holder or a swabprocessing insert. The swab holder or swab processing insert can betapered or angled to allow a single sample processing element toaccommodate all types of swabs by allowing swabs with different amountsof fiber, or that are wound to different levels of tightness, to be heldsecurely within the holder or insert. Most preferably, the swab holderor swab processing insert securely holds the swab to provide stability.In one embodiment, the sample collection device is dry and sterile. Inanother embodiment, a dry, sterile sample collection device is moistenedbefore use with a sterile liquid. The moistening solution can be acarrier solution to assist in the collection of material. Alternately,the moistening solution can be a transport solution for the sampling ofmicrobes and viruses. A suitable viral transport medium is 5% tryptosephosphate broth, 0.5% bovine serum albumin, and antibiotics inphosphate-buffered saline.

The sample collection step can also be incorporated into a diagnosticdevice. In one embodiment, the sample is collected by a point of carediagnostic device. In a preferred embodiment, the point of carediagnostic device is a lateral flow immunoassay. Patents concerning theuse of such devices include U.S. Pat. Nos. 5,079,142; 5,591,645;5,601,986; 5,622,871; 6,228,660; and 7,109,042. Foreign applicationsinclude PCT GB 88-00322. All patents and applications are incorporatedby reference in their entirety.

In some instances, samples may be collected from individuals repeatedlyover a longitudinal period of time (e.g., once a day, once a week, oncea month, biannually or annually). Obtaining numerous samples from anindividual over a period of time can be used to verify results fromearlier detections and/or to identify an alteration as a result of, forexample, drug treatment. Samples can be obtained from humans ornon-humans. Preferably, samples are obtained from humans.

Analysis

In the present invention, a specimen of nasal mucus, secretion, ortissue is collected and analyzed using one or more analytical techniquesincluding enzymatic technique, ELISA, fluorometric technique, massspectrography, HPLC, GLC, PCR, and other similar techniques. The presentinvention also includes methods of diagnosing a disease by analyzingnucleic acids in a nasal specimen by nucleic acid detection methods suchas, but are not limited to polymerase chain reaction (PCR), ligase chainreaction (LCR), strand displacement amplification (SDA), self-sustainedsequence replication (3SR), array based tests, and TAQMAN. A number oftemplate dependent processes are available to amplify the targetsequences of interest present in a sample. One of the best knownamplification methods is the polymerase chain reaction (PCR) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159.

Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) is a process for amplifying one ormore desired specific nucleic acid sequences found in a nucleic acid.Because large amounts of a specific sequence may be produced by thisprocess, it is used for improving the efficiency of cloning DNA ormessenger RNA and for amplifying a target sequence to facilitatedetection thereof.

PCR involves a chain reaction for producing, in exponential quantitiesrelative to the number of reaction steps involved, at least one specificnucleic acid sequence given (a) that the ends of the required sequenceare known in sufficient detail that oligonucleotides can be synthesizedwhich will hybridize to them, and (b) that a small amount of thesequence is available to initiate the chain reaction. The product of thechain reaction would be a discrete nucleic acid duplex with terminicorresponding to the ends of the specific primers employed.

Any source of nucleic acid, in purified or non purified form, can beutilized as the starting nucleic acid or acids, provided it contains oris suspected of containing the specific nucleic acid sequence desired.Thus, the process may employ, for example, DNA or RNA, includingmessenger RNA, which DNA or RNA may be single stranded or doublestranded. In addition, a DNA-RNA hybrid which contains one strand ofeach may be utilized. A mixture of any of these nucleic acids may alsobe employed, or the nucleic acid produced from a previous amplificationreaction herein using the same or different primers may be so utilized.The specific nucleic acid sequence to be amplified may be only afraction of a larger molecule or can be present initially as a discretemolecule, so that the specific sequence constitutes the entire nucleicacid. It is not necessary that the sequence to be amplified be presentinitially in a pure form; it may be a minor fraction of a complexmixture, such as a portion of the β-globin gene contained in whole humanDNA or a portion of nucleic acid sequence due to a particularmicroorganism which organism might constitute only a very minor fractionof a particular biological sample. The starting nucleic acid may containmore than one desired specific nucleic acid sequence which may be thesame or different. Therefore, it is useful not only for producing largeamounts of one specific nucleic acid sequence, but also for amplifyingsimultaneously more than one different specific nucleic acid sequencelocated on the same or different nucleic acid molecules.

The nucleic acid or acids may be obtained from any source, for example,from plasmids such as pBR322, from cloned DNA or RNA, or from naturalDNA or RNA from any source, including bacteria, yeast, viruses, andhigher organisms such as plants or animals. DNA or RNA may be extractedfrom blood, tissue material such as chorionic villi or amniotic cells.

It will be understood that the word primer as used hereinafter may referto more than one primer, particularly in the case where there is someambiguity in the information regarding the terminal sequence(s) of thefragment to be amplified. For instance, in the case where a nucleic acidsequence is inferred from protein sequence information a collection ofprimers containing sequences representing all possible codon variationsbased on degeneracy of the genetic code will be used for each strand.One primer from this collection will be 100% homologous with the end ofthe desired sequence to be amplified.

An appropriate agent may be added for inducing or catalyzing the primerextension reaction and the reaction is allowed to occur under conditionsknown in the art. The inducing agent may be any compound or system whichwill function to accomplish the synthesis of primer extension products,including enzymes. Suitable enzymes for this purpose may include, forexample, E. coli DNA polymerase I, Klenow fragment of E. coli DNApolymerase I, T4 DNA polymerase, other available DNA polymerases,reverse transcriptase, and other enzymes, including heat-stable enzymes,which will facilitate combination of the nucleotides in the propermanner to form the primer extension products which are complementary toeach nucleic acid strand. Generally, the synthesis can be initiated atthe 3′ end of each primer and proceed in the 5′ direction along thetemplate strand, until synthesis terminates, producing molecules ofdifferent lengths. There may be inducing agents, however, which initiatesynthesis at the 5′ end and proceed in the other direction, using thesame process as described above.

The newly synthesized strand and its complementary nucleic acid strandform a double-stranded molecule which can be used in the succeedingsteps of the process. In the next step, the strands of thedouble-stranded molecule may be separated to provide single-strandedmolecules. New nucleic acid may be synthesized on the single-strandedmolecules. Additional inducing agent, nucleotides and primers may beadded if necessary for the reaction to proceed under the conditionsprescribed above. Again, the synthesis would be initiated at one end ofthe oligonucleotide primers and would proceed along the single strandsof the template to produce additional nucleic acid. After this step,half of the extension product would consist of the specific nucleic acidsequence bounded by the two primers. The steps of strand separation andextension product synthesis can be repeated as often as needed toproduce the desired quantity of the specific nucleic acid sequence. Theamount of the specific nucleic acid sequence produced would accumulatein an exponential fashion. After the appropriate length of time haspassed to produce the desired amount of the specific nucleic acidsequence, the reaction may be halted by inactivating the enzymes in anyknown manner or separating the components of the reaction.

Amplification is useful when the amount of nucleic acid available foranalysis is very small, as, for example, in the prenatal diagnosis ofsickle cell anemia using DNA obtained from fetal cells. Amplification isparticularly useful if such an analysis is to be done on a small sampleusing non-radioactive detection techniques which may be inherentlyinsensitive, or where radioactive techniques are being employed butwhere rapid detection is desirable.

Any known techniques for nucleic acid (e.g., DNA and RNA) amplificationcan be used with the assays described herein. Preferred amplificationtechniques are the polymerase chain reaction (PCR) methodologies whichcomprise solution PCR and in situ PCR.

The invention is not limited to the use of straightforward PCR. A systemof nested primers may be used for example. Other suitable amplificationmethods known in the field can also be applied such as, but not limitedto, ligase chain reaction (LCR), strand displacement amplification(SDA), self-sustained sequence replication (3SR), array based test, andTAQMAN.

As used herein “amplification” may refer to any in vitro method forincreasing the number of copies of a nucleic acid sequence with the useof a DNA polymerase. Nucleic acid amplification results in theincorporation of nucleotides into a DNA molecule or primer therebyforming a new DNA molecule complementary to a DNA template. The newlyformed DNA molecule and its template can be used as templates tosynthesize additional DNA molecules. As used herein, one amplificationreaction may consist of many rounds of DNA replication. DNAamplification reactions include, for example, polymerase chain reactions(PCR). One PCR reaction may consist of 5-100 “cycles” of denaturation,annealing, and synthesis of a DNA molecule.

Fluorescence Microscopy

Some embodiments of the invention include fluorescence microscopy for adetection of a biological substance in a nasal specimen. Fluorescencemicroscopy enables the molecular composition of the structures beingobserved to be identified through the use of fluorescently-labeledprobes of high chemical specificity such as antibodies. It can be doneby directly conjugating a fluorophore to a protein and introducing thisback into a cell. Fluorescent analogue may behave like the nativeprotein and can therefore serve to reveal the distribution and behaviorof this protein in the cell. Along with NMR, infrared spectroscopy,circular dichroism and other techniques, protein intrinsic fluorescencedecay and its associated observation of fluorescence anisotropy,collisional quenching and resonance energy transfer are techniques forprotein detection.

The naturally fluorescent proteins can be used as fluorescent probes.The jellyfish aequorea victoria produces a naturally fluorescent proteinknown as green fluorescent protein (GFP). The fusion of thesefluorescent probes to a target protein enables visualization byfluorescence microscopy and quantification by flow cytometry. Withoutlimiting the scope of the present invention, some of the probes are asfollowing:

Labels:

Sensitivity and safety (compared to radioactive methods) of fluorescencehas led to an increasing use for specific labeling of nucleic acids,proteins and other biomolecules. Besides fluorescein, other fluorescentlabels cover the whole range from 400 to 820 nm. By way of example only,some of the labels are, fluorescein and its derivatives,carboxyfluoresceins, rhodamines and their derivatives, atto labels,fluorescent red and fluorescent orange: Cy3/Cy5 alternatives, lanthanidecomplexes with long lifetimes, long wavelength labels—up to 800 nm, DYcyanine labels, and phycobili proteins.

Conjugates:

Antibody conjugates can be generated with specificity for virtually anyepitope and are therefore, applicable to imaging a wide range ofbiomolecules. By way of example only, some of the conjugates are,isothiocyanate conjugates, streptavidin conjugates, and biotinconjugates.

Enzyme Substrates:

By way of example only, some of the enzyme substrates are fluorogenicand chromogenic substrates.

Micro- and Nanoparticles:

By way of example only, some of the fluorochromes are: FITC (greenfluorescence, excitation/emission=506/529 nm), rhodamine B (orangefluorescence, excitation/emission=560/584 nm), and nile blue A (redfluorescence, excitation/emission=636/686 nm). Fluorescent nanoparticlescan be used for various types of immunoassays. Fluorescent nanoparticlesare based on different materials, such as, polyacrylonitrile, andpolystyrene etc.

Molecular Rotors:

Fluorescent molecular rotors are sensors of microenvironmentalrestriction that become fluorescent when their rotation is constrained.Few examples of molecular constraint include increased dye(aggregation), binding to antibodies, or being trapped in thepolymerization of actin.

IEF-Markers:

IEF (isoelectric focusing) is an analytical tool for the separation ofampholytes, mainly proteins. An advantage for IEF-Gel electrophoresiswith fluorescent IEF-marker is the possibility to directly observe theformation of gradient. Fluorescent IEF-marker can also be detected byUV-absorption at 280 nm (20° C.).

Any or all of these fluorescent probes can be used for the detection ofbiological substances in the nasal mucus. A peptide library can besynthesized on solid supports and, by using coloring receptors,subsequent dyed solid supports can be selected one by one. If receptorscannot indicate any color, their binding antibodies can be dyed. Themethod can not only be used on protein receptors, but also on screeningbinding ligands of synthesized artificial receptors and screening newmetal binding ligands as well. Automated methods for HTS and FACS(fluorescence activated cell sorter) can also be used. A FACS machineoriginally runs cells through a capillary tube and separate cells bydetecting their fluorescent intensities.

Immunoassays

Some embodiments of the invention include immunoassay for a detection ofa biological substance in a nasal specimen. In immunoblotting like thewestern blot of electrophoretically separated proteins a single proteincan be identified by its antibody. Immunoassay can be competitivebinding immunoassay where analyte competes with a labeled antigen for alimited pool of antibody molecules (e.g. radioimmunoassay, EMIT).Immunoassay can be non-competitive where antibody is present in excessand is labeled. As analyte antigen complex is increased, the amount oflabeled antibody-antigen complex may also increase (e.g. ELISA).Antibodies can be polyclonal if produced by antigen injection into anexperimental animal, or monoclonal if produced by cell fusion and cellculture techniques. In immunoassay, the antibody may serve as a specificreagent for the analyte antigen.

Without limiting the scope and content of the present invention, some ofthe types of immunoassays are, by way of example only, RIAs(radioimmunoassay), enzyme immunoassays like ELISA (enzyme-linkedimmunosorbent assay), EMIT (enzyme multiplied immunoassay technique),microparticle enzyme immunoassay (MEIA), LIA (luminescent immunoassay),and FIA (fluorescent immunoassay). These techniques can be used todetect biological substances in the nasal specimen. Theantibodies—either used as primary or secondary ones—can be labeled withradioisotopes (e.g. 125I), fluorescent dyes (e.g. FITC) or enzymes (e.g.HRP or AP) which may catalyse fluorogenic or luminogenic reactions.

EMIT (Enzyme Multiplied Immunoassay Technique):

EMIT is a competitive binding immunoassay that avoids the usualseparation step. A type of immunoassay in which the protein is labeledwith an enzyme, and the enzyme-protein-antibody complex is enzymaticallyinactive, allowing quantitation of unlabelled protein.

ELISA (Enzyme Linked Immunosorbent Assay):

Some embodiments of the invention include ELISA to detect biologicalsubstances in the nasal specimen. ELISA is based on selective antibodiesattached to solid supports combined with enzyme reactions to producesystems capable of detecting low levels of proteins. It is also known asenzyme immunoassay or EIA. The protein is detected by antibodies thathave been made against it, that is, for which it is the antigen.Monoclonal antibodies are often used.

The test may require the antibodies to be fixed to a solid surface, suchas the inner surface of a test tube, and a preparation of the sameantibodies coupled to an enzyme. The enzyme may be one (e.g.,β-galactosidase) that produces a colored product from a colorlesssubstrate. The test, for example, may be performed by filling the tubewith the antigen solution (e.g., protein) to be assayed. Any antigenmolecules present may bind to the immobilized antibody molecules. Theantibody-enzyme conjugate may be added to the reaction mixture. Theantibody part of the conjugate binds to any antigen molecules that werebound previously, creating an antibody-antigen-antibody “sandwich”.After washing away any unbound conjugate, the substrate solution may beadded. After a set interval, the reaction is stopped (e.g., by adding 1N NaOH) and the concentration of colored product formed is measured in aspectrophotometer. The intensity of color is proportional to theconcentration of bound antigen.

ELISA can also be adapted to measure the concentration of antibodies, inwhich case, the wells are coated with the appropriate antigen. Thesolution (e.g., serum) containing antibody may be added. After it hashad time to bind to the immobilized antigen, an enzyme-conjugatedanti-immunoglobulin may be added, consisting of an antibody against theantibodies being tested for. After washing away unreacted reagent, thesubstrate may be added. The intensity of the color produced isproportional to the amount of enzyme-labeled antibodies bound (and thusto the concentration of the antibodies being assayed).

Radioimmunoassay:

Some embodiments of the invention include radioimmunoassays to detectbiological substances in the nasal specimen. Radioactive isotopes can beused to study in vivo metabolism, distribution, and binding of smallamount of compounds. Radioactive isotopes of ¹H, ¹²C, ³¹P, ³²S, and ¹²⁷Iin body are used such as ³H, ¹⁴C, ³²P, ³⁵S, and ¹²⁵I.

In receptor fixation method in 96 well plates, receptors may be fixed ineach well by using antibody or chemical methods and radioactive labeledligands may be added to each well to induce binding. Unbound ligands maybe washed out and then the standard can be determined by quantitativeanalysis of radioactivity of bound ligands or that of washed-outligands. Then, addition of screening target compounds may inducecompetitive binding reaction with receptors. If the compounds showhigher affinity to receptors than standard radioactive ligands, most ofradioactive ligands would not bind to receptors and may be left insolution. Therefore, by analyzing quantity of bound radioactive ligands(or washed-out ligands), testing compounds' affinity to receptors can beindicated.

The filter membrane method may be needed when receptors cannot be fixedto 96 well plates or when ligand binding needs to be done in solutionphase. In other words, after ligand-receptor binding reaction insolution, if the reaction solution is filtered through nitrocellulosefilter paper, small molecules including ligands may go through it andonly protein receptors may be left on the paper. Only ligands thatstrongly bound to receptors may stay on the filter paper and therelative affinity of added compounds can be identified by quantitativeanalysis of the standard radioactive ligands.

Fluorescence Immunoassays:

Some embodiments of the invention include fluorescence immunoassays fora detection of a biological substance in a nasal specimen. Fluorescencebased immunological methods are based upon the competitive binding oflabeled ligands versus unlabeled ones on highly specific receptor sites.

The fluorescence technique can be used for immunoassays based on changesin fluorescence lifetime with changing analyte concentration. Thistechnique may work with short lifetime dyes like fluoresceinisothiocyanate (FITC) (the donor) whose fluorescence may be quenched byenergy transfer to eosin (the acceptor). A number of photoluminescentcompounds may be used, such as cyanines, oxazines, thiazines,porphyrins, phthalocyanines, fluorescent infrared-emitting polynucleararomatic hydrocarbons, phycobiliproteins, squaraines and organo-metalliccomplexes, hydrocarbons and azo dyes.

Fluorescence based immunological methods can be, for example,heterogenous or homogenous. Heterogenous immunoassays comprise physicalseparation of bound from free labeled analyte. The analyte or antibodymay be attached to a solid surface. The technique can be competitive(for a higher selectivity) or noncompetitive (for a higher sensitivity).Detection can be direct (only one type of antibody used) or indirect (asecond type of antibody is used). Homogenous immunoassays comprise nophysical separation. Double-antibody fluorophore-labeled antigenparticipates in an equilibrium reaction with antibodies directed againstboth the antigen and the fluorophore. Labeled and unlabeled antigen maycompete for a limited number of anti-antigen antibodies.

Some of the fluorescence immunoassay methods include simple fluorescencelabeling method, fluorescence resonance energy transfer (FRET), timeresolved fluorescence (TRF), and scanning probe microscopy (SPM). Thesimple fluorescence labeling method method can be used forreceptor-ligand binding, enzymatic activity by using pertinentfluorescence, and as a fluorescent indicator of various in vivophysiological changes such as pH, ion concentration, and electricpressure. TRF is a method that selectively measures fluorescence of thelanthanide series after the emission of other fluorescent molecules isfinished. TRF can be used with FRET and the lanthanide series can becomedonors or acceptors. In scanning probe microscopy, in the capture phase,for example, at least one monoclonal antibody is adhered to a solidphase and a scanning probe microscope is utilized to detectantigen/antibody complexes which may be present on the surface of thesolid phase. The use of scanning tunneling microscopy eliminates theneed for labels which normally is utilized in many immunoassay systemsto detect antigen/antibody complexes.

Nuclear Magnetic Resonance (NMR)

Some embodiments of the invention include NMR for detection of abiological substance in a nasal specimen. NMR spectroscopy is capable ofdetermining the structures of biological macromolecules like proteinsand nucleic acids at atomic resolution. In addition, it is possible tostudy time dependent phenomena with NMR, such as intramolecular dynamicsin macromolecules, reaction kinetics, molecular recognition or proteinfolding. Heteronuclei like ¹⁵N, ¹³C and ²H, can be incorporated inproteins by uniform or selective isotopic labeling. Additionally, somenew information about structure and dynamics of macromolecules can bedetermined with these methods.

X-Ray Crystallography

Some embodiments of the invention include X-ray crystallography fordetection of a biological substance in a nasal specimen. X-raycrystallography is a technique in which the pattern produced by thediffraction of X-rays through the closely spaced lattice of atoms in acrystal is recorded and then analyzed to reveal the nature of thatlattice. This generally leads to an understanding of the material andmolecular structure of a substance. The spacings in the crystal latticecan be determined using Bragg's law. X-ray diffraction is commonlycarried out using single crystals of a material, but if these are notavailable, microcrystalline powdered samples may also be used which mayrequire different equipment.

Fluorescence Spectroscopy

Some embodiments of the invention include fluorescence spectroscopy fordetection of a biological substance in a nasal specimen. By way ofexample only, conventional fluorometry is measurement of emission lightintensities at defined wavelengths for a certain emission maxima of afluorophore. Total fluorometry is a collection of data for a continuumof absorption as well as emission wavelengths. Fluorescence polarizationis when polarized light is used for excitation and binding offluorochrome-labeled antigens to specific antibodies. Line narrowingspectroscopy is low-temperature solid-state spectroscopy that derivesits selectivity from the narrow-line emission spectra.

Time-dependent fluorescence spectroscopy comprises time-resolvedmeasurements containing more information than steady-state measurements,since the steady-state values represent the time average oftime-resolved determinations. It is a single photon timing techniquewhere the time between an excitation light pulse and the first photonemitted by the sample is measured.

Matrix Assisted Laser Desorption Ionization Time-of-Flight MassSpectrometry (MALDI TOF-MS)

Some embodiments of the invention include MALDI TOF-MS for detection ofa biological substance in a nasal specimen. MALDI TOF-MS providesaccurate mass determinations and primary sequence information. Improvedmass resolution in MALDI TOF-MS can be obtained by the utilization of asingle-stage or a dual-stage reflectron (RETOF-MS). In the reflectronmass spectrum, the isotopic multiplet is well resolved producing a fullwidth half maximum (FWHM) mass resolution of about 3400. Massresolutions up to 6000 (FWHM) can be obtained for peptides up to about3000 Da with RETOF-MS Enhancing the mass resolution can also increasethe mass accuracy when determining the ion's mass.

Both linear and reflectron MALDI-TOF-MS can be utilized for molecularweight determinations of molecular ions and enzymatic digests leading tostructural information of proteins. These digests are typically massanalyzed with or without purification prior to molecular weightdeterminations. Varieties of methodologies have been developed to obtainprimary sequence information for proteins and peptides utilizing MALDITOF-MS. Two different approaches can be taken. The first method is knownas protein ladder sequencing and can be employed to produce structurallyinformative fragments of the analyte prior to insertion into the TOFmass spectrometer and subsequent analysis. The second approach utilizesthe phenomenon of metastable ion decay that occurs inside the TOF massspectrometer to produce sequence information.

The ladder sequencing with TOF-MS consists of either a time-dependent orconcentration-dependent chemical degradation from either the N- orC-terminus of the protein/peptide into fragments, each of which differsby one amino acid residue. The mixture is mass analyzed in a singleMALDI-TOF-MS experiment with mass differences between adjacent massspectral peaks corresponding to a specific amino acid residue. The orderof occurrence in the mass spectrum defines the sequence of amino acidsin the original protein/peptide.

Post-source decay with RETOF-MS MALDI is an ionization technique thatproduces intact protonated pseudomolecular ion species. A significantdegree of metastable ion decay occurs after ion acceleration and priorto detection. The ion fragments produced from the metastable ion decayof peptides and proteins typically include both neutral molecule losses(such as water, ammonia and portions of the amino acid side chains) andrandom cleavage at peptide bonds. In-source decay with linear TOF-MS isan alternative approach to RETOF-MS for studying metastable ion decay ofMALDI generated ions. Primary structural information for peptides andproteins can be obtained by this method. Coherent mass spectral peakscan be produced from these metastable decayed ions giving rise tosignificant structural information for peptides and proteins.

Surface-Enhanced Laser Desorption Ionization-Time of Flight (SELDI-TOF)

Some embodiments of the invention include SELDI TOF-MS for detection ofa biological substance in a nasal specimen. This technique utilizesstainless steel or aluminum-based supports, or chips, engineered withchemical (hydrophilic, hydrophobic, pre-activated, normal-phase,immobilized metal affinity, and cationic or anionic) or biological(antibody, antigen binding fragments (e.g. scFv), DNA, enzyme, orreceptor) bait surfaces of 1-2 mm in diameter. These varied chemical andbiochemical surfaces allow differential capture of proteins based on theintrinsic properties of the proteins themselves. Solubilized tissue orbody fluids in volumes as small as 0.1 μl can be directly applied tothese surfaces, where proteins with affinities to the bait surface maybind. Following a series of washes to remove non-specifically or weaklybound proteins, the bound proteins are laser desorbed and ionized for MSanalysis. Masses of proteins ranging from small peptides of less than1000 Da up to proteins of greater than 300 kDa can be calculated basedon time-of-flight. As mixtures of proteins may be analyzed withindifferent samples, a unique sample fingerprint or signature may resultfor each sample tested. Consequently, patterns of masses rather thanactual protein identifications can be produced by SELDI analysis. Thesemass spectral patterns can be used to differentiate patient samples fromone another, such as diseased from normal.

UV-Vis

Some embodiments of the invention include optical absorptionspectroscopy (UV/VIS) for detection of a biological substance in a nasalspecimen. UV/VIS provides light absorption data which helps in thedetermination of concentration of macromolecules such as, proteins, DNA,nucleotides etc. Organic dyes can be used to enhance the absorption andto shift the absorption into the visible range (e.g. coomassie bluereagents). Resonance raman spectroscopy (RRS) can be used to studymolecular structure and dynamics. RRS helps in investigating specificparts of macromolecules by using different excitation wavelengths.

Liquid Chromatography (LC)

Some embodiments of the invention include LC for a detection ofbiological substance in a nasal specimen. Examples of LC are but notlimited to, affinity chromatography, gel filtration chromatography,anion exchange chromatography, cation exchange chromatography, diodearray—LC and high performance liquid chromatography (HPLC).

Gel filtration chromatography separates proteins, peptides, andoligonucleotides on the basis of size. Molecules may move through a bedof porous beads, diffusing into the beads to greater or lesser degrees.Smaller molecules may diffuse further into the pores of the beads andtherefore move through the bed more slowly, while larger molecules mayenter less or not at all and thus move through the bed more quickly.Both molecular weight and three dimensional shapes contribute to thedegree of retention. Gel filtration chromatography may be used foranalysis of molecular size, for separations of components in a mixture,or for salt removal or buffer exchange from a preparation ofmacromolecules.

Affinity chromatography is the process of bioselective adsorption andsubsequent recovery of a compound from an immobilized ligand. Thisprocess allows for the specific and efficient purification of manydiverse proteins and other compounds. Ion exchange chromatographyseparates molecules based on differences between the overall charges ofthe proteins. It can be used for the purification of protein,oligonucleotides, peptides, or other charged molecules.

HPLC can be used in the separation, purification and detection ofbiological substances in the nasal mucus. Crude tissue extracts may beloaded directly onto the HPLC system and mobilized by gradient elution.Rechromatography under the identical conditions is an option if furtherpurification is warranted or necessary. Reversed phase chromatography(RPC) can be utilized in the process of protein structure determination.HPLC may be coupled with MS. The HPLC method described in Henkin et al.,New Frontiers in Immunobiology, 2000, pp. 127-152, is incorporatedherein in its entirety.

Size-exclusion chromatography (SEC) and ion-exchange chromatography(IEC) can be used for separation and purification of biologically activeproteins, such as enzymes, hormones, and antibodies. In liquid affinitychromatography (LAC), interaction may be based on binding of the proteindue to mimicry of substrate, receptor, etc. The protein may be eluted byintroducing a competitive binding agent or altering the proteinconfiguration which may facilitate dissociation. A procedure that can beused in the separation of membrane proteins is the use of nonionicdetergents, such as Triton X-100 (Polyethylene glycol tert-octylphenylether), or protein solubilization by organic solvents with IEC.

Diode array detector-liquid chromatography (DAD-LC) provides complete,multiple spectra for each HPLC peak, which, by comparison, can provideindication of peak purity. These data can also assign presence of tyr,trp, phe, and possibly others (his, met, cys) and can quantitate theseamino acids by 2nd derivative or multi-component analysis. By apost-column derivatization, DAD-LC can also identify and quantitate cys,his and arg in individual peptides. Thus, it is possible to analyze for6 of the 20 amino acids of each separated peptide in a single LC run,and information can be obtained about presence or absence of these aminoacids in a given peptide in a single step. This is assisted by knowingthe number of residues in each peptide.

Electrophoresis

Some embodiments of the invention include electrophoresis for detectionof a biological substance in a nasal specimen. Electrophoresis can begel electrophoresis or capillary electrophoresis.

Gel Electrophoresis:

Gel electrophoresis is a technique that can be used for the separationof proteins. During electrophoresis, macromolecules are forced to movethrough pores when an electrical current is applied. Their rate ofmigration through the electric field depends on strength of the field,size and shape of the molecules, relative hydrophobicity of the samples,and on an ionic strength and temperature of a buffer in which themolecules are moving. After staining, the separated macromolecules ineach lane can be seen in a series of bands spread from one end of thegel to the other. Using this technology it is possible to separate andidentify protein molecules that differ by as little as a single aminoacid. Also, gel electrophoresis allows determination of crucialproperties of a protein such as its isoelectric point and approximatemolecular weight. Electrofocusing or isoelectric focusing is a techniquefor separating different molecules by their electric charge differences(if they have any charge). It is a type of zone electrophoresis thattakes advantage of the fact that a molecule's charge changes as the pHof its surroundings changes.

Capillary Electrophoresis:

Capillary electrophoresis is a collection of a range of separationtechniques which may involve the application of high voltages acrossbuffer filled capillaries to achieve separations. The variations includeseparation based on size and charge differences between analytes (termedcapillary zone electrophoresis (CZE) or free solution CE (FSCE)),separation of neutral compounds using surfactant micelles (micellarelectrokinetic capillary chromatography (MECC) or sometimes referred toas MEKC) sieving of solutes through a gel network (capillary gelelectrophoresis, GCE), separation of cations (or anions) based onelectrophoretic mobility (capillary isotachophoresis, CITP), andseparation of zwitterionic solutes within a pH gradient (capillaryisoelectric focusing, CIEF). Capillary electrochromatography (CEC) canbe an associated electrokinetic separation technique which involvesapplying voltages across capillaries filled with silica gel stationaryphases. Separation selectivity in CEC can be a combination of bothelectrophoretic and chromatographic processes. Many of the CE separationtechniques rely on the presence of an electrically induced flow ofsolution (electroosmotic flow, EOF) within the capillary to pump solutestowards the detector.

Arrays

Some embodiments of the invention include arrays for detection of abiological substance in a nasal specimen. Arrays involve performingparallel analysis of multiple samples against known protein targets. Thedevelopment of various microarray platforms can enable and acceleratethe determination of protein abundance, localization, and interactionsin a cell or tissue. Microarrays provide a platform that allowsidentification of protein interaction or function against acharacterized set of proteins, antibodies, or peptides. Protein-basedchips array proteins on a small surface and can directly measure thelevels of proteins in tissues using fluorescence-based imaging. Proteinscan be arrayed on either flat solid phases or in capillary systems(microfluidic arrays), and several different proteins can be applied tothese arrays. In addition to the use of antibodies as array probes,single-stranded oligonucleotides, whose specificity is optimized by invitro elution (aptamers), offer a viable alternative. Nonspecificprotein stains can be then used to detect bound proteins.

Arrays include, but are not limited to, bead arrays, bead based arrays,bioarrays, bioelectronic arrays, cDNA arrays, cell arrays, DNA arrays,gene arrays, gene expression arrays, frozen cell arrays, genome arrays,high density oligonucleotide arrays, hybridization arrays,microcantilever arrays, microelectronic arrays, multiplex DNAhybridization arrays, nanoarrays, oligonucleotide arrays,oligosaccharide arrays, planar arrays, protein arrays, solution arrays,spotted arrays, tissue arrays, exon arrays, filter arrays, microarrays,small molecule microarrays, suspension arrays, theme arrays, tilingarrays, and transcript arrays.

Sensors

Some embodiments of the invention include sensors for detection of abiological substance in a nasal specimen. Sensors can be used for bothin vivo and in vitro detection. Sensors can be chemical sensors, opticalsensors, and biosensors. Chemical sensors are miniaturized analyticaldevices which may deliver real-time and online information on thepresence of specific compounds or ions in complex samples. Opticalsensors are based on measurement of either intrinsic optical propertiesof analytes, or of optical properties of indicator dyes or labeledbiomolecules attached to solid supports. Biosensors can be affinitybiosensor based on capabilities of enzymes to convert substrates intoproducts or catalytic biosensors. Biosensors detect antibody and analytecomplexes using a variety of physical methods. Some biosensors measurethe change in surface charge that occurs when analyte is bound toantibodies or other binding agents, which in turn are bound to asurface. Other biosensors use binding agents attached to a surface andmeasure a change in a physical property of the support, other thansurface charge, upon binding of analyte. Some biosensor techniques use aspecific property of a labeled binding agent or antigen to produce ameasurable change.

Methods for Identifying Proteins from a Library Screen

Protein identification methods by way of example only includelow-throughput sequencing through Edman degradation, mass spectrometrytechniques, peptide mass fingerprinting, de novo sequencing, andantibody-based assays. The protein quantification assays includefluorescent dye gel staining, tagging or chemical modification methods(i.e. isotope-coded affinity tags (ICATS), combined fractional diagonalchromatography (COFRADIC)). The purified protein may also be used fordetermination of three-dimensional crystal structure, which can be usedfor modeling intermolecular interactions. Common methods for determiningthree-dimensional crystal structure include x-ray crystallography andNMR spectroscopy. Detailed below are a few of the methods foridentifying proteins in the present invention.

Protein Sequencing:

N-terminal sequencing aids in the identification of unknown proteins,confirms recombinant protein identity and fidelity (reading frame,translation start point, etc.), aids the interpretation of NMR andcrystallographic data, demonstrates degrees of identity betweenproteins, or provide data for the design of synthetic peptides forantibody generation, etc. N-terminal sequencing utilises the Edmandegradative chemistry, sequentially removing amino acid residues fromthe N-terminus of the protein and identifying them by reverse-phaseHPLC. Sensitivity can be at the level of 100 s femtomoles and longsequence reads (20-40 residues) can often be obtained from a few 10 spicomoles of starting material. Pure proteins (>90%) can generate easilyinterpreted data, but insufficiently purified protein mixtures may alsoprovide useful data, subject to rigorous data interpretation.N-terminally modified (especially acetylated) proteins cannot besequenced directly, as the absence of a free primary amino-groupprevents the Edman chemistry. However, limited proteolysis of theblocked protein (e.g. using cyanogen bromide) may allow a mixture ofamino acids to be generated in each cycle of the instrument, which canbe subjected to database analysis in order to interpret meaningfulsequence information. C-terminal sequencing is a post-translationalmodification, affecting the structure and activity of a protein. Variousdisease situations can be associated with impaired protein processingand C-terminal sequencing provides an additional tool for theinvestigation of protein structure and processing mechanisms.

Proteome Analyses:

Proteomics can be identified primarily by computer search algorithmsthat assign sequences to a set of empirically acquired mass/intensitydata which are generated from conducting electrospray ionization (ESI),matrix-assisted laser desorption/ionization (MALDI-TOF), orthree-dimensional quadrupole ion traps on the protein of interest.

Diagnosis

The identification and analysis of biological substances as disclosedherein has numerous therapeutic and diagnostic applications. Clinicalapplications include, for example, detection of disease, distinguishingdisease states to inform prognosis, selection of therapy, and/orprediction of therapeutic response, disease staging, identification ofdisease processes, prediction of efficacy of therapy, monitoring ofpatients trajectories (e.g., prior to onset of disease), prediction ofadverse response, monitoring of therapy associated with efficacy andtoxicity, and detection of recurrence.

Measuring a concentration of the biological substance can aid in thediagnosis of a course of a disease. For example, the diabetic state of apatient who was previously diagnosed with diabetes can be determined bymonitoring the nasal secretions of the patient for insulin. A biologicalsubstance, for example, growth factor may be one that is specific forthe patient's specific disease. Alternatively, a panel of two or morespecific or nonspecific growth factors may be monitored. Theconcentrations of either an individual factor or several factors, in thebiological sample of the patient may be affected by the disease.

The presence or increase or decrease of biological substances'concentration allows the physician or veterinarian to predict the courseof the disease or the efficacy of treatment regimes. If, for example, apatient who had a certain type of disease, which was treated,subsequently exhibits an increase in the concentration of biologicalsubstances that is associated with that disease, the physician orveterinarian can predict that the patient may have progression of thedisease in the future or predict a higher risk of fatality in thepatient. In addition, the amount of biological substances may bepredictive of the outcome of the patient, e.g., how well certainchemotherapeutic agents may act.

One aspect of the present invention is a method of diagnosing a diseaseby obtaining a specimen of nasal secretion, detecting a biologicalsubstance in the specimen, and diagnosing the disease wherein thediagnosis is based on the detection of the biological substance, andwherein the biological substance is not related to a respiratorydisease. In one embodiment leprosy is diagnosed by detection ofantibodies against leprosy causing pathogen for example, mycobacteriumleprae. In one embodiment hepatitis, such as hepatitis A, B, C, D, E,and G, is diagnosed by detection of antibodies against hepatitis causingvirus. In some embodiments, the biological substances include insulin orinsulin receptors for a diagnosis of diabetes. In some embodiments, thebiological substance is p53 for a diagnosis of cancer.

Some embodiments of the invention include diagnosing diabetes bydetecting insulin or insulin receptor in the nasal specimen. Table 6depicts detection and measurement of human insulin concentration innasal mucus as compared to insulin concentration in blood plasma andsaliva. Table 7 depicts the detection and measurement of human insulinreceptor concentration in nasal mucus as compared to the insulinreceptor concentration in plasma and saliva. The appearance of insulinor insulin receptors in nasal mucus reflects either their synthesis innasal serous glands or response to a physiological and/or pathologicalphenomena. The presence of insulin or insulin receptors in nasal mucusoffers a non-invasive method for the diagnosis of diabetes and otherdisorders of carbohydrate metabolism.

Some embodiments of the invention include diagnosing cancer by detectingcaspase in the nasal specimen. Cysteine-dependent aspartate-specificproteases (caspases) are a family of proteases that cleave theirsubstrates at aspartic acid (D)-X bonds. 14 mammalian caspases have beenidentified. Caspase-2, -3, -6, -7, -8, -9 and -10 are major players inthe execution phase of apoptosis, whereas caspase-1, -4, -5, and -11 areinvolved in cytokine processing associated with inflammation. Caspase 3,also known as CPP32, cleaves and activates a variety of proteins such assterol regulatory element binding proteins (SREBPs). Caspase-3 alsocleaves poly (ADP-ribose) polymerase (PARP) at the onset of apoptosisand amyloid β precursory protein (APP) which is associated with neuronaldeath in Alzheimer's disease. Caspase 3 is activated by graszyne β,ADAF-1, caspase 9 and caspases 6, 8 and 10. This substance is one of theapoptotic substances found during the apoptotic process. Table 10illustrates a comparison between the detection of caspase 3 in nasalmucus as well as saliva. The presence of caspase in nasal mucus is about13% of that in saliva and reflects the magnitude of the apoptoticprocess. The presence of caspase in nasal mucus indicates the activityof cellular death in nasal mucus and shows that cancer can be diagnosedby detecting caspase in the nasal specimen.

Some embodiments of the invention include diagnosing cancer, taste lossor smell loss by detecting tumor necrosis factor α (TNFα) in the nasalspecimen. TNFα is a 17 KD cleavage product mediated by TNFα convertingenzyme which interacts with two distinct TNFα receptors (I, II) on thecell surface. TNFα is upregulated in many pathological processesinvolving inflammation and oncological processes such as rheumatoidarthritis, refractory bronchial asthma, liver disease, cancer and inpatients with taste and smell loss. It is also called cachectin and isproduced by many normal and tumor cells in response to a wide variety ofstimuli including viruses, bacteria, parasites, cytokines and mitogens.Both the transmembrane and the soluble secreted forms of TNFα arebiologically active. TNFα is an extremely pleotropic cytokine due to theubiquity of its receptors, its ability to activate multiple signaltransduction pathways and its ability to induce or suppress theexpression a large number of genes.

Detecting the levels of TNFα in nasal mucus as disclosed herein makesthe diagnosis of a disease possible on a clinical basis since obtainingcellular diagnosis through tissue biopsy can not only be invasive butalso can be difficult and at times dangerous. Table 11 illustratesdetection and measurement of TNFα in nasal mucus and saliva in 75subjects. Results indicate that TNFα in nasal mucus is about 30 timeshigher than in saliva. These data suggest that various cancers can bediagnosed by measurements of TNFα in nasal mucus and their treatment canbe monitored by following its concentration in nasal mucus. Since levelsof TNFα may also reflect the inflammatory aspects of disease processesinducing it, use of anti TNFα drugs through nasal administration reflecta method of treating these various disease processes. Concentrations ofTNFα in nasal mucus in patients with smell loss can be greater than forexample, 5000 times that found in normal subjects thereby reflecting itsfunction as a “death protein” indicator of excessive apoptosis as in itsrole in cancer.

Monitoring the levels of TNFα in nasal mucus may help in diagnosis ofarterial venous malfunction (AVM). AVM is normally diagnosed by MRI butdetection and measurement of TNFα in nasal mucus provides a non-invasivemethod of diagnosing AVM. Monitoring the level of TNFα can help indiagnosing the progression or stage of AVM or the susceptibility of thesubject towards AVM.

Some embodiments of the invention include diagnosing cancer, taste lossor smell loss by detecting tumor necrosis factor receptor I (TNFR I) inthe nasal specimen. TNF receptor I (TNFRI) is one of the two cellularreceptors upon which TNFα operates. It is one of the prototypic membersof the TNF receptor super family members designated TNFRSF I α. Indisease processes associated with increased TNFα activity TNFR I may beupregulated. Its presence in nasal mucus can reflect the activity ofmany inflammatory, oncological and other pathological processes,including taste and smell dysfunction. Table 12 illustrates detectionand measurement of TNFR I in 47 subjects. Results indicate that TNFR Iin nasal mucus is about 16 times its concentration in saliva and itsconcentration is significantly increased over that found in plasma, redblood cells, or urine. Thus the detection of TNFR I in the nasalspecimen can be used to establish clinical diagnoses of excessiveapoptosis and can be used as a treatment modality in inhibitingpathological apoptosis.

TNFR II is the other of the two cellular receptors upon which TNFαoperates. TNFR II is one of the high-affinity receptors for TNFα and isone of the prototypic members of the TNF super family members designatedTNFSF I β. TNFR II may be upregulated in many inflammatory andoncological disease processes. It may also be solubilized and haveproperties similar to TNFR I. Table 13 illustrates detection andmeasurement of TNFR II in 47 subjects. Results indicate that TNFR II innasal mucus is about 24 times its concentration in saliva and itsconcentration in nasal mucus is significantly higher than found inplasma, rbcs, or urine. The results reflect that detection of TNFR II innasal specimen can provide a non invasive method of diagnosing variouspathological processes related to TNFR II.

TNF related apoptosis-inducing ligand (TRAIL) is also known as apo-2ligand and TNFSF-10. It is a Type II transmembrane protein with acarboxy terminal extracellular domain that exhibits homology to otherTNF super family members. Among TNF super family members TRAIL is themost homologous Fas ligand, sharing 28% amino acid sequence identity intheir extracellular domain. Human TRAIL shares 65% amino acid sequenceidentity with mouse TRAIL. TRAIL reflects the terminal protein in theapoptotic sequence. Table 14 in the examples illustrates detection andmeasurement of TRAIL in saliva and nasal mucus in normal subjects and inpatients with smell loss. Results indicate that TRAIL in nasal mucus isabout 5 times higher than in saliva and both are significantly higherthan in blood, rbcs or urine. The results reflect that detection ofTRAIL in nasal mucus can provide a non invasive method of diagnosingvarious diseases related to TRAIL. The methods of the present inventioninclude treatment of diseases by modulating these elevatedconcentrations by use of anti-TRAIL drugs or agents. In someembodiments, the treatment is preferably by nasal administration.

Some embodiments of the invention include diagnosing disease bydetecting interleukin in the nasal specimen. Interleukin 2 (IL2) is aT-cell growth factor that is produced by T-cells following activation bymitogins or antigens and it stimulate growth and differentiation of βcells, natural killer (NK) cells, lymphocyte killer (LAK) cells,monocytes/macrophages and oligodendrocytes. At the amino acid sequencelevel there can be about 50-90% homology between species. Interleukin 3(IL 3), also known as mast cell growth factor, is a pleitropic factorproduced primarily by activated T cells. It can stimulate proliferationand differentiation of pluripotent hematopoetic stem cells as well asvarious lineage committed progenitors. Mature human and mouse IL 3 shareabout 29% amino acid sequence homology. Table 18 in the examplesillustrates detection and measurement of IL 3 in both human saliva andnasal mucus. Levels of IL 3 in nasal mucus were found to be about ½ theconcentration in saliva but both levels were higher than that found inplasma, rbcs or urine. IL 3 present in nasal mucus provides a noninvasive method of diagnosing various diseases related to IL3. Themethods of the present invention include treatment of diseases bymodulating the concentration of IL3 with drugs. In some embodiments, thetreatment is by nasal administration.

Some embodiments of the invention include diagnosing disease bydetecting endostatin in the nasal specimen. Endostatin is a 20 KD Gterminal fragment of collagen XVII. Its function is as an antiangiogenicsubstance or angiogenic antagonist. It is a naturally occurring proteinwhich has been used as an anti-cancer agent to inhibit blood vesselgrowth and spread of any form of cancer. Table 19 in the examplesillustrates detection and measurement of endostatin in plasma, urine,saliva and nasal mucus in 15 subjects. Endostatin levels in nasal mucuswere 5 times higher than in saliva but 7% that found in plasma. On thebasis of endostatin/protein, levels of nasal mucus are about 14% thatfound in plasma. Presence of endostatin in nasal mucus indicates anon-invasive method of detection of endostatin in nasal mucus and itsuse in diagnosing various diseases. The methods of the present inventioninclude treatment of diseases by modulating the concentration ofendostatin with drugs. In some embodiments, the treatment is by nasaladministration.

Some embodiments of the invention include diagnosing disease bydetecting erythropoietin in the nasal specimen. Erythropoietin (EPO) isa 30 KD glycosylated protein produced primarily by the kidney. It is theprincipal factor that regulates erythropoesis. Production of EPO by thekidney cell is increased in response to hyposmia or anemia. The cDNA forEPO has been cloned from many species. Mature proteins from variousspecies are highly conserved exhibiting greater than 80% amino acidsequence homology. Table 20 in the examples illustrates detection andmeasurement of EPO in plasma, urine, saliva and nasal mucus. EPO was notfound in urine or saliva. The level of EPO in nasal mucus was found tobe between 1.1 and 4.5 times higher than in plasma. Presence of EPO innasal mucus illustrates a non-invasive method of detection of EPO innasal mucus and its use in diagnosing various diseases. The diagnosiscan further lead to treatment of disease by modulating the concentrationof EPO with drugs. In some embodiments, the treatment is by nasaladministration.

Some embodiments of the invention include diagnosing disease bydetecting bone morphogenic protein in the nasal specimen. Bonemorphogenic protein BMP I, also known as procollagen C-proteinase (PCP)is a zinc protease of the astacin family. BMP I/PCP plays a key role information of extracellular matrix (KCM) by connecting precursor proteinsinto their mature and functional form. Precursor proteins identified assubstrates for BMP I/PCP include collagens, biglycan, laminin S, dentinmatrix protein I and lysyl oxidase. Table 21 in the examples illustratesdetection and measurement of BMP I in plasma, urine, saliva and nasalmucus in 20 subjects. BMP I was found in plasma but not in urine, salivaor nasal mucus.

Some embodiments of the invention include diagnosing disease bydetecting brain-derived neurotrophic factor in the nasal specimen.Brain-derived neurotrophic factor (BDNF) is a member of the NGF familyof neurotrophic factors, BDNF, NGF, NT-3 and NT 4/5. BDNF is requiredfor differentiation and survival of specific subpopulations in bothcentral and peripheral nervous systems. High levels of BDNF expressionhave been found in hippocampus, cerebellum, fetal eye and placenta.Table 22 in the examples illustrates detection and measurement of BDNFin plasma, urine, saliva and nasal mucus in 20 subjects. BDNF was foundin plasma and nasal mucus but not in urine or saliva. Levels of BDNF inplasma were higher than in nasal mucus. The results indicate that nasalmucus is a repository of the family of nerve growth factors and theconcentration of BDNF as shown in Table 22, may help understand bothphysiology and pathology of neurotrophic factors related to growth andhomeostasis of cells in the nasal cavity as well as reporting on thepresence of these factors in the systemic circulation. Presence of BDNFin nasal mucus illustrates a non-invasive method of detection of BDNF innasal mucus and its use in diagnosing various diseases. The diagnosiscan further lead to treatment of diseases by modulating theconcentration of BDNF with drugs. In some embodiments, the treatment isby nasal administration.

Some embodiments of the invention include diagnosing disease bydetecting ciliary neurotrophic factor in the nasal specimen. Ciliaryneurotrophic factor (CNTF) is structurally related to IL-6, IL-11, L1F,CLC and OSM. CNTF is a trophic factor for embryonic chick ciliaryparasympathetic neurons in culture. CNTF is also a survival factor foradditional numerous cell types including dorsal root ganglion sensoryneurons, sympathetic ganglion neurons, embryonic motor neurons, majorpelvic ganglion neurons and hippocampal neurons. Table 23 in theexamples illustrates detection and measurement of CNTF in plasma, urine,saliva and nasal mucus in 19 subjects. Levels of CNTF in plasma andnasal mucus were found to be similar but lower in saliva. Presence ofCNTF in nasal mucus illustrates a non-invasive method of detection ofCNTF in nasal mucus and its use in diagnosing various diseases. Thediagnosis can further lead to treatment of diseases by modulating theconcentration of CTNF with drugs. In some embodiments, the treatment isby nasal administration.

Some embodiments of the invention include diagnosing disease bydetecting granulocyte macrophage growth factor in the nasal specimen.Granulocyte macrophage growth factor (GM-CSF) is a 22 KD mononerichematopoetic cytokine that is characterized as a growth factor thatsupports the in vitro colony formation of granulocyte macrophageprogenitors. It is produced by a number of different cell typesincluding activated T cells, B cells, macrophages, mast cells,endothelial cells and fibroblasts, in response to cytokines or immuneand inflammatory stimuli. GM-CSF is species specific. Table 24 in theexamples illustrates detection and measurement of GM-CSF in plasma,urine, saliva and nasal mucus in 16 subjects. The results provide anon-invasive method for the detection of GM-CSF in nasal mucus. Levelsin nasal mucus were found to be over 6 times that found in plasma. Thedetection of GM-CSF in nasal mucus provides a non invasive method ofdiagnosing various diseases related to GM-CTF. The methods of thepresent invention include treatment of diseases by modulating theconcentrations of GM-CSF by use of drugs. In some embodiments, thetreatment is by nasal administration.

Some embodiments of the invention include diagnosing disease bydetecting hepatocyte growth factor in the nasal specimen. Hepatocytegrowth factor (HGF), also known as hepatopoeitin A, is a mitogenicprotein for a variety of cell types including endothelial and epithelialcells, melanocytes and keratinocytes. It is identical to scatter factor,a fibroblast-derived soluble factor that promotes the dissociation ofepithelial and vascular endothelial cell colonies in monolayes culturesby stimulating cell migration. Table 25 in the examples illustratesdetection and measurement of HGF in plasma, urine, saliva and nasalmucus in 17 subjects. Concentrations of HGF in nasal mucus were found tobe higher than that found in either plasma or urine. These resultssuggest that HGF may be synthesized in the serous glands of the nose fora specific mechanism involved with nasal homeostasis as well as amechanism involved with systemic cell migration. The results provide anon-invasive method for the detection of HGF in nasal mucus. Thedetection of HGF in nasal mucus provides a non invasive method ofdiagnosing various diseases related to human physiology and pathology.The diagnosis further leads to a treatment of diseases by modulating theconcentrations of HGF by use of drugs.

Some embodiments of the invention include diagnosing disease bydetecting platelet derived growth factor in the nasal specimen. Plateletderived growth factor (PDGF) family is a group of disulfide-linkeddimeric proteins which act mainly on connective tissue. This family mayconsist of four homodimeric proteins, PDGF-AA, PDGF-BB, PDGF-CC andPDGF-DD and one heterodimeric protein, PDGF-AB. The technique of ELISAmeasurement used is associated with the ability of PDGF to stimulateincorporation of 3H-thymidine in quiescent NRGR-3 T 3 fibroblastis.Table 26 in the examples illustrates detection and measurement of PDGFin human plasma, urine, saliva and nasal mucus in 18 subjects.Concentrations of PDGF expressed per mg protein were found to be higherin saliva and nasal mucus than in plasma. These results suggest thatPDGF may be synthesized in the serous glands of the nose for a specificmechanism involved with nasal homeostasis. The results provide anon-invasive method for the detection of PDGF in nasal mucus. Thedetection of PDGF in nasal mucus provides a non invasive method ofdiagnosing various diseases related to human physiology and pathology.The diagnosis can provide treatment of diseases by modulating theconcentrations of PDGF by use of drugs.

Some embodiments of the invention include diagnosing taste loss or smellloss by detecting carbonic anhydrase in the nasal specimen. Carbonicanhydrase is a zinc-containing enzyme and at least twenty carbonicanhydrase variants, called “isozymes” have been identified. CarbonicAnhydrase VI (CA VI) is a 36 KD zinc metalloglycoprotein. Its synthesisin nasal mucus may take place in nasal serous glands (in the oralparotid glands). It can act as a taste bud growth factor in the oralcavity and as an olfactory receptor growth factor in the nasal cavity.It can also act on taste bud and olfactory receptor stem cells to inducegrowth and development of the entire panoply of cell types for the tastebuds and olfactory epithelium. Its decreased synthesis may induce bothloss of taste and smell. Its resumed synthesis may return cell growth tonormal. Treatment which increases synthesis of CA VI may involve severalcomplex processes including increasing zinc-cofactor concentration.Administration of zinc ion to some patients who are either zincdeficient or who may have metabolic processes which inhibit zincincorporation into the protein, may have their taste and smell functionimproved through this treatment. Since the carbohydrates in this proteinare part of its function, any process that repairs glycoproteinincorporation into this protein may also therapeutically be effective inrestoring taste and smell function.

Table 27 in the examples illustrates decrease in CA VI in patients withsmell and taste loss. Table 28 in the examples illustrates loss of smellfunction by disease etiology with respect to measurements of CA VIconcentration in nasal mucus. Results indicate that patients with postinfluenza hyposmia hypogeusia (PIHH), allergic rhinitis and postanesthesia have significantly decreased CA VI concentrations in nasalmucus. These results provide a method for the detection and measurementof CA VI in nasal mucus as an index of smell and taste loss and itscontinual measurement during treatment of these disorders in order tomonitor efficacy of therapy. The detection of CA VI in nasal mucusprovides a non invasive method of diagnosing various diseases related tohuman physiology and pathology. The diagnosis can further lead totreatment of diseases by modulating the concentrations of CA VI by useof drugs

Some embodiments of the invention include diagnosing a disease bydetecting cAMP and cGMP in the nasal specimen. Table 29 in the examplesillustrates detection and measurement of cAMP and cGMP in saliva and innasal mucus in normal subjects. Table 30 in the examples illustratescomparison of the measurement of cAMP and cGMP in normal subjects withthe patients with taste and smell loss. Results indicated that patientswith smell loss had decreased levels of cAMP in their nasal mucus. Theseresults indicate that cAMP in nasal mucus can be an index of smell lossand that its secretion may be inhibited in smell loss. The resultsprovide a non-invasive method for the detection of cAMP and cGMP innasal mucus.

Table 31 in the examples illustrates detection and measurement of cAMPand cGMP secretion in nasal mucus in patients with graded severity ofsmell loss (anosmia<Type I hyposmia<Type II hyposmia from most severe toleast severe). Data indicates that as degree of smell loss increased,levels of cAMP in nasal mucus decreased. These data confirm therelationship between cAMP secretion in nasal mucus and degree of smellloss. Results also indicate that there was less significant differencebetween cGMP in nasal mucus in normal subjects or in patients withhyposmia. However, the concentration of cGMP in saliva is essentiallysimilar to that of cAMP, phenomena different from that observed in othertissues.

The ability to smell and, in part, the ability to taste or to obtainflavor from food is regulated by the olfactory nerve system. Theolfactory nerve system is complex and interconnected with severalsystems in the brain. Olfactory receptors located in the nose arespecialized bipolar neurons with cilia protruding into the mucouscovering the epithelium. The axons of the bipolar neurons are packedinto bundles that form connections in the olfactory bulb in the brain.The olfactory bulbs contain a rich supply of neurotransmitters andneuromodulators. Chemosensory dysfunctions are usually described by thefollowing terms: ageusia (absence of taste), hypogeusia, (diminishedsensitivity of taste), dysgeusia (distortion of normal taste), anosmia(absence of smell), hyposmia (diminished sense of smell), and dysosmia(distortion of normal smell).

Treatment with drugs which increase cAMP secretion (e.g., thephosphodiesterase theophylline or cilostazol) increases nasal mucus cAMPconcentration and are associated with increases in smell function. Thus,cAMP measurements are critical to monitor both loss of smell functionand changes in smell function following treatment. The detection of cAMPand cGMP in nasal mucus provides a non-invasive method of diagnosingvarious diseases related to human physiology and pathology. The methodsof the present invention include treatment of diseases by modulating theconcentrations of cAMP and cGMP by use of drugs or agents. The method oftreatment is preferably by nasal administration.

Some embodiments of the invention include diagnosing a disease bydetecting nitric oxide in the nasal specimen. Nitric oxide (NO) is apletrophic-signaling molecule implicated in diverse biological processesincluding inhibition of platelet aggregation, regulation ofneurotransmission, vasodilation, immune responses and inflammation. NOis synthesized from arginine and O₂ by three nitric oxide synthase(NOS), enzymes endothelial NOS (eNOS), neuronal NOS (nNOS), andinducible NOS (iNOS). Each enzyme isoform is expressed in a variety oftissues and cell types. While eNOS and nNOS generally exhibitconstitutive expression and are involved in physiological signaling andcellular maintenance functions, iNOS expression may be induced byinflammatory stimuli and may be associated with both normal andpathological immune responses. Table 32 in the examples illustratesdetection and measurement of NO in human saliva and nasal mucus. NO wasfound to be present is in both saliva and nasal mucus and its meanconcentration in saliva were 21% lower in patients than in normalsubjects whereas in nasal mucus mean levels were 25% lower in patients.Treatment which increases cAMP in nasal mucus and improves smellfunction may be mirrored by increases in nasal mucus NO.

Some embodiments of the invention include diagnosing a disease bydetecting insulin-like growth factor I in the nasal specimen.Insulin-like growth factor I (IGF 1), also known as somatomedin Cbelongs to the family of insulin-like growth factors that arestructurally homologous to proinsulin. IGF 1 is a potent mitogenicfactor that mediates growth-promoting activities of growth hormonepostnatally. IGF 1 also promotes growth during embryonic growth anddifferentiation. Table 34 in the examples illustrates detection andmeasurement of IGF 1 in human saliva and nasal mucus in 26 subjects.Results show that IGF 1 concentration in nasal mucus was significantlygreater than in saliva. Results indicate that the measurement of nasalmucus IGF 1 can be used as an index of human physiology and pathology.The detection of IGF 1 in nasal mucus provides a non-invasive method ofdiagnosing various diseases related to human physiology and pathology.The diagnosis can further help in treatment of diseases by modulatingthe concentrations of IGF 1 by use of drugs.

Some embodiments of the invention include diagnosing a disease bydetecting endoglin in the nasal specimen. Endoglin, also known as CD105, is a type 1 integral membrane glycoprotein and is an accessoryreceptor for TGF-β super family ligands. Endoglin is expressed onvascular endothelial cells, chrondrocytes and syncytiotrophoblasts ofterm placenta. It is also found on activated monocytes, mesenchymol stemcells and leukemic cells of lymphoid and myeloid lineages. Table 39illustrates detection and measurement of endoglin in the nasal mucus.Results indicate that the measurement of nasal mucus endoglin can beused as an index of human physiology and pathology. The detection ofendoglin in nasal mucus provides a non invasive method of diagnosingvarious diseases related to human physiology and pathology.

Some embodiments of the invention include diagnosing a disease bydetecting fibroblast growth factor (FGF) in the nasal specimen. FGFacidic is a member of the FGF family of mitogenic peptides. Unlike othermembers of the family, it lacks signal peptides. FGF is apparentlysecreted by mechanisms other than the classical protein secretionpathways. There are approximately 23 distinct members of this family.The nucleotide sequence of human FGF acidic is well known and it is a155 amino acid protein. FGF mediates cellular responses by binding toand activating a family of four receptor tyrosine kinases. FGF isinvolved in wound healing as it binds heparin. It promotes endothelialcell proliferation by physical organization of endothelial cells intotubes. It promotes angiogenesis and stimulates the proliferation offibroblasts that give rise to granulation tissue. It is a more potentangiogenic factor than either VFGF or PDGF. It acts on PC 12 cells;these cells also respond to NGF in a similar manner. Low levels of FGFhave been found in blood of patients with depression. Acidic FGF wasmeasured in blood plasma, urine, saliva and nasal mucus in 13 subjects.No FGF was found in any sample of plasma, urine or saliva. FGF wasmeasured in three samples of the nasal mucus or in 23 percent of thesubjects. Values ranged from 8-44 pg/ml with a mean±SEM of 24±13 pg/ml.These results suggest that FGF is present in nasal mucus and may be partof a feedback mechanism involving nasal cavity and brain since FGF issynthesized in the brain.

The diagnosis of the disease as disclosed herein can be used to enableor assist in the pharmaceutical drug development process for therapeuticagents. The analysis can be used to diagnose disease for patientsenrolling in a clinical trail. The diagnosis can indicate the state ofthe disease of patients undergoing treatment in clinical trials, andshow changes in the state during the treatment. The diagnosis candemonstrate the efficacy of a treatment, and can be used to stratifypatients according to their responses to various therapies.

The methods of the present invention can be used to evaluate theefficacy of treatments over time. For example, sample of nasalsecretions can be obtained from a patient over a period of time as thepatient is undergoing treatment. The DNA from the different samples canbe compared to each other to determine the efficacy of the treatment.Also, the methods described herein can be used to compare the efficaciesof different therapies and/or responses to one or more treatments indifferent populations (e.g., different age groups, ethnicities, familyhistories, etc.).

In preferred embodiment, at least one step of the methods of the presentinvention is performed using a computer as depicted in FIG. 2. FIG. 2illustrates a computer for implementing selected operations associatedwith the methods of the present invention. The computer 200 includes acentral processing unit 201 connected to a set of input/output devices202 via a system bus 203. The input/output devices 202 may include akeyboard, mouse, scanner, data port, video monitor, liquid crystaldisplay, printer, and the like. A memory 204 in the form of primaryand/or secondary memory is also connected to the system bus 203. Thesecomponents of FIG. 2 characterize a standard computer. This standardcomputer is programmed in accordance with the invention. In particular,the computer 200 can be programmed to perform various operations of themethods of the present invention.

The memory 204 of the computer 200 may store a detection/diagnosismodule 205. In other words, the detection/diagnosis module 205 canperform the operations associated with step 102, 103, and 104 of FIG. 1.The term “detection/diagnosis module” used herein includes, but notlimited to, analyzing one or more biological substances, identifying thebiological substance, and diagnosing the disease after theidentification. The executable code of the detection/diagnosis module205 may utilize any number of numerical techniques to perform thediagnosis.

Examples of Biological Substances

Various substances that can be diagnosed by the methods of the presentinvention include, by way of example only, proteins, carbohydrates,lipids, hormones (e.g., leptin, ghrelin) in control of appetite,cholesterol and other lipids and lipid carrying proteins in control oflipid metabolism, growth factors (e.g., hepatic growth factor,granulocyte colony growth factor, brain derived neurotrophic factor),liver enzymes (SGOT, SGPT) therapeutic and recreational drugs of abuse,trace metals [either excess as in toxicity (e.g., lead, mercury,arsenic) or in deficiency diseases involving zinc, copper, magnesium]and most other substances found in plasma, erythrocytes, urine andsaliva. Each metabolite in nasal mucus may reflect both physiologicaland pathological changes in human body metabolism specific to eachmetabolite and may reflect the manner in which nasal mucus providesinformation both on human body metabolism such as provided by plasma,erythrocytes, urine and saliva or information relatively unique to nasalmucus.

The methods of the present invention include PCR to enable detectionand/or characterization of specific nucleic acid sequences associatedwith infectious diseases, genetic disorders or cellular disorders.Various infectious diseases can be diagnosed by the presence in clinicalsamples of specific DNA sequences characteristic of the causativemicroorganism.

Infectious organisms may comprise viruses, (e.g., single stranded RNAviruses, single stranded DNA viruses, human immunodeficiency virus(HIV), hepatitis A, B, and C virus, herpes simplex virus (HSV),cytomegalovirus (CMV) Epstein-Barr virus (EBV), human papilloma virus(HPV)), parasites (e.g., protozoan and metazoan pathogens such asPlasmodia species, Leishmania species, Schistosoma species, Trypanosomaspecies), bacteria (e.g., Mycobacteria, M. tuberculosis, Salmonella,Chlamydia, Neisseria, Streptococci, E. coli, Staphylococci, C. psittaciand C. pecorum), fungi (e.g., Acremonium; Absidia (e.g., Absidiacorymbifera). Aspergillus (e.g., Aspergillus clavatus, Aspergillusflavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger,Aspergillus terreus, Aspergillus versicolor, etc), Blastomyces (e.g.,Blastomyces dermatitidis, etc), Candida (e.g., Candida albicans, Candidaglabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candidaparapsilosis, Candida stellatoidea, Candida tropicalis, Candida utilis,etc.), Cladosporium (e.g., Cladosporium trichoides, etc), Coccidioides(e.g., Coccidioides immitis, etc), Cryptococcus (e.g., Cryptococcusneoformans, etc), Cunninghamella (e.g., Cunninghamella elegans, etc),Dermatophyte, Exophiala (e.g., Exophiala dermatitidis, Exophialaspinifera, etc), Epidermophyton (e.g., Epidermophyton floccosum, etc),Fonsecaea (e.g., Fonsecaea pedrosoi, etc), Fusarium (e.g., Fusariumsolani, etc), Geotrichum (e.g., Geotrichum candiddum, etc), Histoplasma(e.g., Histoplasma capsulatum var. capsulatum, etc), Malassezia (e.g.,Malassezia furfur, etc), Microsporum (e.g., Microsporum canis,Microsporum gypseum, etc), Mucor, Paracoccidioides (e.g.,Paracoccidioides brasiliensis, etc), Penicillium (e.g., Penicilliummarneffei, etc), Phialophora, Pneumocystis (e.g., Pneumocystis carinii,etc), Pseudallescheria (e.g., Pseudallescheria boydii, etc), Rhizopus(e.g., Rhizopus microsporus var. rhizopodiformis, Rhizopus oryzae, etc),Saccharomyces (e.g., Saccharomyces cerevisiae, etc), Scopulariopsis,Sporothrix (e.g., Sporothrix schenckii, etc), Trichophyton (e.g.,Trichophyton mentagrophytes, Trichophyton rubrum, etc), Trichosporon(e.g., Trichosporon asahii, Trichosporon cutaneum, etc).), Pneumocystiscarinii, and prions.

Other examples of biological substances, includes, but is not limitedto, colony stimulating factors (1, 2, 3, GM, α, β, γ, and the like), Bcell factors (B cell growth factor and the like), T cell factors,protein A, suppressive factor of allergy, suppressor factors, cytotoxicglycoprotein, immunocytotoxic agents, immunotoxins, lymphotoxins,cachectin, oncostatins, tumor inhibitory factors, albumin,α-1-antitrypsin, apolipoprotein, erythroid potentiating factors,erythropoietin, factor VII, factor VIII(c), factor IX, hemopoietin-1,kidney plasminogen activator, tissue plasminogen activator, urokinase,pro-urokinase, streptokinase, lipocortin, lipomodulin, macrocortin, lungsurfactant protein, protein C, protein 5, C-reactive protein, renininhibitors, collagenase inhibitors, superoxide dismutase, growthhormone, osteogenic growth factors, atrial naturetic factor, auriculin,atriopeptin, bone morphogenic protein, calcitonin, calcitonin precursor,calcitonin gene-related peptide, cartilage inducing factor, connectivetissue activator protein, fertility hormones (follicle stimulatinghormone, luteinizing hormone, human chorionic gonadotropin), growthhormone releasing factor, osteogenic protein, insulin, proinsulin, nervegrowth factor, parathyroid hormone and analogues, parathyroid hormoneantagonists, relaxin, secretin, somatomedin C, somatostatin andsomatostatin analogues, inhibin, adrenocoricotrophic hormone, glucagon,vasoactive intestinal polypeptide, gastric inhibitory peptide, motilin,cholecystokinin, pancreatic polypeptide, gastrin releasing peptide,corticotropin releasing factor, thyroid stimulating hormone, growthinhibitory factors, vaccine antigens including antigens of HTLV-I, II,HTLV-III/LAV/HIV (AIDS virus), cytomegalovirus, hepatitis A, B, andnon-A/non-B, herpes simplex virus-I, herpes simplex virus II, malaria,pseudorabies, retroviruses, feline leukemia virus, bovine leukemiavirus, transmissible gastroenteritis virus, infectious bovinerhinotracheitis, parainfluenza, influenza, rotaviruses, respiratorysyncytial virus, varicella zoster virus, epstein-barr virus, pertussis,and anti-infective antibodies including monoclonal and polyclonalantibodies to gram negative bacteria, pseudomonas, endotoxin, tetanustoxin, and other bacterial or viral or other infectious organisms.

In addition to naturally-occurring allelic forms of growth inhibitoryfactor, the present invention also embraces other inhibitory factorproducts such as polypeptide analogs of inhibitory factor. Such analogsinclude fragments of inhibitory factor. Other examples of biologicalsubstances, includes, substances that are associated with cancer (eitheractive or remission) and/or with reaction to transplantation (eithertissue acceptance or rejection).

We compared levels of many biologic substances in nasal mucus with thosein blood, urine and saliva using a 96 plate colorimetric ELISA assay.Most of these substances are detectable in nasal mucus, sometimes atlevels that are significantly higher compared to other biologic fluids.These results indicate that, while the specific mechanisms for thepresence of these varied substances in nasal mucus are unclear, theirpresence reflects unique indicators of both human physiology anddisease.

Methods of the present invention include methods for the detection ofthe following biologic substances in nasal mucus: agouti relatedprotein, alpha fetoprotein (AFP), brain derived neurotrophic factor(BDNF), bone morphogenetic protein-2 (BMP-2), ciliary neurotrophicfactor (CNTF), thymus and activation-regulated chemokines (CCL17/TARC)CC chemokines, cystatin, D-dimer, E selectin, endoglin, epidermal growthfactor, (EGF), endothelial nitric oxide synthase, (eNOS), FAS ligand,fibroblastic growth factor basic (FGF basis), granulocyte macrophagecolony stimulating factor (GM-CSF), hepatocyte growth factor (HGF),inducible nitric oxide synthase (iNOS), insulin-like growth factor 1(IGF-1), interferon alpha (INF-α), interferon beta (INF-β), interferongamma (INF-γ), interferon omega (INF-ω), intracellular adhesion molecule1 (ICAM-1), interleukin-1 (IL-1), interleukin-1 receptor (IL-1receptor), interleukin-2 (IL-2), interleukin-2 receptor (IL-2 receptor),interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-15 (IL-15),interleukin-17 (IL-17), interleukin-18 (IL-18), keratinocyte growthfactor (KGF), L-selectin, leptin, leukemia inhibitor factor (LIF),matrix metalloproteinase 1 (MMP-1), migrating inhibitory factor (MIF),nerve growth factor (NGF), P selectin, placental growth factor (PlGF),platelet derived growth factor-AA (PDGF-AA), platelet derived growthfactor-BB (PDGF-BB), pro-B type natiuretic peptide, receptor forabdominal glycation end product (RAGE), stem cell factor (SCF),substance P, triggering receptor expressed on myeloid cells (TREM-1),transforming growth factor alpha (TGF-alpha), transforming growth factorbeta (TGF-beta), tumor necrosis factor (TNF), tumor necrosis factorreceptor 1 (TNF-R1), tumor necrosis factor receptor 2 (TNF-R2),TNF-related apoptosis-inducing ligand (TRAIL), vascular cell adhesionmolecule 1 (VCAM1), vascular endothelial growth factor C (VEGF-C),vascular growth factor D (VEGF-D), vascular endothelia growth factorreceptor 1 (VEGFR1), or vascular endothelia growth factor receptor 2(VEGFR2). Levels of these biologic substances are directly or indirectlyassociated with numerous disease processes or condition and theirconcentration in nasal mucus may increase or decrease during the courseof disease progression. Detection of a biologic substance in nasal mucuscan be used as a diagnostic method for identifying a disease andascertaining the stage of the disease process. Levels of some biologicsubstances may change prior to the appearance of disease symptoms andmay therefore represent a means by which a disease or condition may bedetected early, providing the opportunity for early treatment includingprophylactic treatment. Additionally, the level of these biologicsubstances may change with the appropriate disease treatment andtherefore, response to treatment can also be used to measure throughnasal mucus levels of these substances. Furthermore, as many of thesesubstances are themselves potent biologic agents, they can beadministered prophylactically or with intent to treat a particulardisease. Nasal mucus monitoring then provides a way to titrate theadministered dosage to achieve the desired therapeutic response and toensure that the patient is receiving a medically adequate dose.

Detection of biological substances can also provide information fromwhich the likelihood of the occurrence of a disease or condition can becalculated. Table 47. This information can be used by a health careprovider in developing a preventative treatment strategy. For example,if detection of a biological substance is linked to an increased risk ofa myocardial infarction, the health care provider can counsel thepatient to make changes to the patient's diet, initiate or intensive anexercise program, prescribe a medication or change medications.Detection of a biological substance can also indicate the likelihoodand/or rate of disease progression. Based on the information currenttreatment can be modified, including adding therapeutic agents ormodalities.

Some embodiments of the invention include diagnosing a disease bydetecting alpha fetoprotein (AFP). AFP is a fetal/tumor associatedprotein that is also a member of the albuminoid super family. AFP canact as carrier protein binding several types of molecules includingsteroids, bilirubin, fatty acids, retinoids and flavanoids. AFP can alsoregulate cell growth and survival. It is secreted by certain cancerssuch as hepatocellular cancer, cancers of the gastrointestinal track,endodermal sinus tumors, neuroblastoma, and hepatoblastoma and hence,increased levels of APF can serve as a biomarker indicating thepresence, hematogenous spread and response to therapy of these cancers.Elevated levels are also seen in multiple gestation as well as in anumber of fetal abnormalities, such as neural tube defects includingspina bifida and anencephaly, abdominal wall defects andAtaxia-telangiotosis. AFP is also elevated in chronic hepatitis Cinfection. In contrast, low levels of maternal serum AFP are associatedwith, Down syndrome and Trisomy 18. Patients with abnormal levels needto undergo detailed obstetric ultrasonography. The information is thenused to decide whether to proceed with amniocentesis. AFP levels may bepredictive of a chromosomally abnormal fetus in the 2^(nd) trimester,fetal Trisomy 18, hepatocellular cancer and gastric cancer with livermetastasis. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting agouti-related protein (AGRP), a neuropeptide and a functionalantagonist of αMSH on melanocortin (MC) 3 and MC4 receptors, both ofwhich are implicated in obesity. It shares sequences and structuralhomology with Agouti protein. Increased levels are associated withobesity and anorexia and hence, AGRP may serve as a biomarker forappetite related diseases. Furthermore, AGRP and related analogs canserve as therapeutic drugs to treat appetite changes and nasal mucusmonitoring may be an ideal way to ascertain drug levels. It wasdiscovered that nasal mucus levels of AGRP to be approximately 10% thatof plasma (p<0.001) and saliva to be approximately 17% of plasma. Table46.

Some embodiments of the invention include diagnosing a disease bydetecting brain-derived neurotrophic factor (BDNF), a member of the NGFfamily of neurotrophic factors. It is required for differentiation andsurvival of specific neuronal subpopulations in both central andperipheral neurons systems. High levels of BDNF are found inhippocampus, cerebellum, fetal eye and placenta. High levels areassociated with medulablastoma and increased itching in eczema.Decreased levels are associated with Huntington's disease, depression,schizophrenia, obsessive-compulsive disorder, Alzheimer's disease, anddementia, and medulloblastoma. Increasing levels of BDNF, additionally,may be predictive of the development of Parkinson's disease. It wasfound that nasal mucus levels of BDNF to be approximately 0.2% of theplasma level (p<0.001) with saliva and urine having undetectable levels.Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting bone morphogenetic protein-2 (BMP-2), a secreted signalingmolecule that composes a subfamily of the TGF-β superfamily. BMP-2 wasoriginally specified as a regulator of cartilage and bone formation.There are at least 20 structurally and functionally active BMPs most ofwhich play roles in embryogenesis and morphogenesis of various bodytissues and organs. Active BMPs are usually heterodimers containing acharacteristic cysteine-knot structure. It is a potent inducer of bonefunction. Increased levels of BMPs are associated with gastric andovarian cancer, malignant melanoma, and chronic renal fibrosis. Table46.

Some embodiments of the invention include diagnosing a disease bydetecting cyclic AMP (cAMP) a growth factor which acts on stem cells intaste buds and olfactory epithelium. If cAMP levels decrease, taste andsmell loss follows. cAMP is also involved as a “second messenger” in allendocrine and many physiological processes.

Some embodiments of the invention include diagnosing a disease bydetecting carbonic anhydrase, an enzyme involved in the functionalprocess of pH control, respiration and sensory function. It acts as agrowth factor for stem cells in taste buds and olfactory epithelium. Ifcarbonic anhydrase levels decrease, taste and smell loss follow.

Some embodiments of the invention include diagnosing a disease bydetecting thymus and activation-regulated chemokines (CCL17/TARC) thatcomprise a subfamily of the chemokines superfamily. They are defined bythe arrangement of the first two of four invariant cysteine residuesfound in all chemokines. In CC chemokines these two cysteines areadjacent. Chemokines bind to multiple 7-transmembrane G protein-coupledCC chemokine receptors. These are small secreted molecules that functionin leukocyte trafficking, recruitment and activation. They also playroles in normal and pathological processes including allergic responses,infection and autoimmune disease, angiogenesis, inflammation and tumorgrowth and metastasis. Increased levels of TARC are associated withHodgkin's lymphoma, Adult T-cell leukemia/lymphoma (ATL), allergicdisease, asthma, atopic dermatitis, systemic lupus erythematosis,angioedema, Trisomy 7 and Paragonimus westermani infection. Thymictumors may secrete an excessive amount of CCL17/TARC and thus may be agood marker for disease activity, either before or after tumortreatment. It was found that nasal mucus levels of TARC wereapproximately 150% of plasma levels, however, the difference was notstatistically significant. Table 46. TARC was not detectable in salivaor urine.

Some embodiments of the invention include diagnosing a disease bydetecting ciliary neurotrophic factor (CNTF), a molecule that isstructurally similar to IL-6, IL-11, CLC and DSM. CNTF is a trophicfactor for embryonic chick ciliary parasympathetic neurons in cultureand is also a survival factor for several neuronal cell types includingdorsal root ganglion sensory neurons, sympathetic ganglion neurons,embryonic motor neurons, major pelvic ganglion neurons and hippocampalneurons. Decreased levels are associated with motor neuron degeneration,amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), andHuntington's disease. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting caspase 3 (CP3), also known CPP32/Yama. Caspases are a familyof cytosolic aspartate-specific cysteine proteases involved in theinitiation and execution of apoptosis. They are expressed as latentzymogens and are activated by auto-proteolytic mechanisms by processingby other proteases (frequently other caspases). Caspases can besubdivided into three function groups—caspase 3 belong to the apoptosisexecution group along with caspase 5 and 7. Some embodiments of theinvention include diagnosing a disease by detecting C reactive protein(CRP). It is a member of the class of acute phase reactants. Levels ofCRP rise dramatically during an acute inflammatory event. The basallevel of CRP is indicative of the general health of a patient. Patientswith elevated basal levels of CRP are at an increased risk for diabetes,hypertension and cardiovascular disease. Colon cancer patients have anelevated level of CRP. Approximately 20% of individual with increasingblood levels of CRP develop chronic obstructive pulmonary disease (COPD)one to eight years later. Monitoring patients for CRP by nasal mucus mayprovide a way to identify patients prone to the disease enablingprophylactic treatment.

Some embodiments of the invention include diagnosing a disease bydetecting cystatin, an inhibitor of cysteine proteases. It is bothintracellular and extracellular. It is a homolog of fetuin, HPRG andkininogen. It regulates protease activity. It is involved in manyphysiological and pathological processes such as tumor invasion andmetastases, inflammation and neurological diseases. Mutations incystatins B & C cause progressive myoclonus epilepsy and a hereditaryform of amyloid angiopathy.

Some embodiments of the invention include diagnosing a disease bydetecting D-dimer, also known as fragment D-dimer, or fibrin degradationfragment. Elevated levels of D-dimer are associated with deep veinthrombosis (DVT), disseminated intravascular coagulation (DIC) andpulmonary embolism. D-dimer levels can also be monitored to assess theeffectiveness of thrombolytic therapy. A preferred embodiment is toassess risk of a repeat thromboembolic event. D-dimer levels in theblood can increase when patients stop anticoagulation therapy fortreatment of thromboembolism. Among patients that stop anticoagulationtherapy, about 15% have abnormal D-dimer levels and are at high risk ofa repeat thromboembolic event. Thus, measurement of D-dimer levels inblood can be used to predict the onset of repeat thromboembolic eventsamong patients who have had a previous event of this type and havestopped anticoagulation therapy. The importance of this finding is thatprior to the discovery of D-dimer in blood and the association withrepeat thromboembolic events, it was impossible to predict the length oftime necessary to continue anticoagulation therapy to inhibit repeatthromboembolism. Now, it may be possible to screen patients for D-dimerby sampling nasal mucus to provide a simple, rapid and low costscreening method for patients at risk for a repeat thromboembolic eventand continue these patients on anticoagulation therapy.

Some embodiments of the invention include diagnosing a disease bydetecting E selectin, also known as CD62E. E selectin, is a celladhesion molecule expressed only on endothelial cells activated bycytokines. Elevated E selectin levels are associated with angiomas,essential hypertension, non-insulin dependent diabetes mellitus,rheumatoid arthritis, and blood forming tumors.

Some embodiments of the invention include diagnosing a disease bydetecting endoglin, a transmembrane glycoprotein. It is an accessoryreceptor for TGF-β superfamily ligands. It is highly expressed onvascular epithelial cells, chondrocytes and syncytiotrophoblasts of termplacenta. It is also found on activated monocytes, mesenchynal stemcells and leukemic cells of lymphoid and myeloid integers. It isincreased in eclampsia and preeclampsia. In a preferred embodiment,endoglin in nasal mucus is used as a prognostic indicator of for thedevelopment of preeclampsia in pregnancy. Preeclampsia occurs in 3-5% ofall pregnancies in the world. It is a significant cause of maternal andfetal mortality. It has recently been demonstrated that circulatinglevels of soluble endoglin are present in serum of pregnant women withpreeclampsia. Table 48. It has also been demonstrated that circulatorylevels of soluble endoglin are significantly elevated in pregnant womenwith preterm preeclampsia. Table 48. Further, circulating levels ofsoluble endoglin are elevated in pregnant women up to 10 weeks prior tothe onset of clinical manifestations of preeclampsia, however, solubleendoglin is not elevated in women who have gestational hypertension orin women who remain normotensive, but deliver growth-restrictedneonates. In this sense, elevated endoglin levels in blood plasma can beused to predict the onset of preeclampsia prior to its clinicalmanifestation.

Levels of endoglin in blood plasma, nasal mucus, saliva and urine in agroup of patients with a variety of clinical disorders were comparedusing a 96 plate colorimetric ELISA assay. Endoglin in plasma rangedfrom 1.1-6.0 ng/ml (2.7±0.2 mean±SEM) whereas in nasal mucus it rangedfrom 0.02-3.0 ng/ml (0.8±0.5); it was not measurable in saliva or urinein any of these patients. Table 49. While levels of endoglin wereapproximately three times higher in plasma than in nasal mucus, thecollection of nasal mucus does not require any invasive procedure and ismore easily performed and accepted by patients than venipuncture.

Some embodiments of the invention include diagnosing a disease bydetecting endostatin, a 20 KD C-terminal fragment of collagen XVIII thatfunctions as an angiogenesis antagonist. Decreased levels are associatedwith cancer including metastatic cancer, gastric ulcers and impairedwound healing.

Some embodiments of the invention include diagnosing a disease bydetecting endothelial nitric oxide synthase (eNOS), also known asconstitutive NOS (cNOS) or type III. eNOS is one of three enzymes thatsynthesize nitric oxide (NO) from L-arginine. Endothelial NOSconstitutively provides a basal release of NO to blood vessels, where NOrelease is involved with regulating vascular function. Decreased levelsof eNOS are associated with heart failure, vascular disease and impairedwound healing. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting epidermal growth factor (EGF), a growth factor derived frommembrane-anchored precursors. EGF promotes proliferation anddifferentiation of mesenchynal and epithelial cells. It is involved insignaling in a wide variety of physiological processes including humancarcinomas. Increased levels are associated with breast cancer, lungcancer and pituitary adenoma. It was found that nasal mucus levels areapproximately 150-fold higher than plasma levels (p<0.0001) and salivalevels approximately 26-fold higher than plasma levels (p<0.001). Table46.

Some embodiments of the invention include diagnosing a disease bydetecting erythropoietin (EPO), a 30 KD heavily glycosylated proteinproduced primarily by the kidney. EPO is the principle factor (hormone)that regulates erythropoiesis (in the bone marrow where is acting as agrowth factor). Its synthesis is increased by hypoxia or anemia.Decreased levels are associated with anemia including chemotherapyinduced anemia, renal failure, severe kidney disease, myelodysplasiasyndromes, aplastic anemia, and uremia. Increased or increasing levelsof EPO are associated with hemodialysis patient response to irontherapy.

Some embodiments of the invention include diagnosing a disease bydetecting FAS-ligand, a 40 KD Type II transmembrane protein belonging tothe TNF superfamily. It is expressed predominantly on activated T cellsand natural killer (NK) cells, whereas FAS is expressed on various celltypes. FAS-ligand/FAS system plays a crucial role in modulating immuneresponses by inducing cell specific apoptosis. Human FAS-ligand shares81% sequence identity with mouse FAS-ligand. Defective Fas mediatedapoptosis may lead to oncogenesis as well as drug resistance in existingtumors. Germline mutation of Fas is associated with autoimmunelymphoproliferative syndrome (ALPS), a childhood disorder of apoptosis.Increased levels of FAS-ligand are associated with leukemia, primarycancers, including lung, colon, pancreas, metastatic cancer,lymphoproliferative diseases, MS, SLE, HIV, hepatitis, and pulmonaryfibrosis. It was discovered that nasal mucus FAS-ligand levels wereapproximately 6% of plasma levels (p<0.001), but approximately 5.5-foldhigher than urine levels (p<0.05). Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting fibroblast growth factor (FGF) acidic, also known as FGF1. FGFacidic is a part of a large family of secreted proteins involved in manyaspects of cellular development including cell proliferation, growth anddifferentiation. FGF acidic acts on several cell types to regulatephysiological functions including angiogenesis, cell growth, patternformation, embryonic development, metabolic regulation, cell migration,neurotrophic effects and tissue repair. Activities are mediated byreceptor tyrosine kinases and are facilitated by hepatic sulfate.Increased levels are associated with bladder cancer, pancreatic cancer,glioblastoma multiforme, meningioma, atheroma, inflammatory arthritis,Crouzon syndrome and spinal cord injuries.

Some embodiments of the invention include diagnosing a disease bydetecting fibroblast growth factor (FGF) basic, also known as bFGF orFGF2, a member of the fibroblast growth factor family. FGF basicstimulates the proliferation of all cells of mesodermal origin and manycells of neuroectodermal, ectodermal, and endodermal origin. FGF basicinduces neuron differentiation, survival, and regeneration. FGF basicalso modulates embryonic development and differentiation. FGF basic mayplay a role in vivo in the modulation of angiogenesis, wound healing andtissue repair, embryonic development and differentiation, and neuronalfunction and neural degeneration. Increased levels of FGF basic areassociated with bladder and pancreatic cancers. Decreased levels of FGFbasic are associated with ischemia cardiac function, Kaposi's sarcomaand brain tumors. It was discovered that FGF basic can be detected innasal mucus, but not in the other bodily fluids. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting granulocyte macrophage growth factor (GM-CSF), a 22 KDmonomeric hematopoietic cytokine characterized initially as a growthfactor that supported in vitro colony formation of granulocytemacrophage progenitors. GM-CSF is produced by several cell typesincluding activated T cells, B cells, macrophages, mast cells,endothelial cells and fibroblasts in response to cytokine or immune andinflammatory stimuli. GM-CSF is species specific. Elevated GM-CSF levelsare associated with asthma, acute and chronic bronchitis, chronicsinusitis, acute myeloblastic leukemia (AML) and melanoma.

Some embodiments of the invention include diagnosing a disease bydetecting hepatocyte growth factor (HGF) also known as hepatopoeitin A.HGF is mitogenic for several cell types including endothelial andepithelial cells, melanocytes and keratinocytes. It is identical toscatter factor, a fibroblast-derived soluble factor that promotesdissociation of epithelial and vascular endothelial cell colonies inmonolayer cultures by stimulating cell migration. It is excreted as aninactive single chain precursor and is converted to the activeheterodimeric form by HGF activation. Elevated levels are associatedwith hepatitis, hepatic failure, cirrhosis, liver regeneration posttransplantation, lung regeneration post transplantation, acute kidneyinjury, kidney disease, myocardial infarction, glioma, and pancreaticcancer.

Inducible nitric oxide synthase (iNOS), also known as type II, is one ofthree enzymes that synthesize nitric oxide (NO) from L-arginine. Nasalmucus levels of iNOS were twice that of plasma levels, but thedifferences were not statistically significant. Table 46. Saliva levelsof iNOS were approximately 60% of nasal mucus levels.

Some embodiments of the invention include diagnosing a disease bydetecting insulin-like growth factor 1 (IGF-1). IGF-1 is mainly secretedby the liver as a result of stimulation by growth hormone (GH) and playsan important role in childhood growth with anabolic effects in adults.Increased levels of IGF-1 are associated with diabetes. Decreased levelsof IGF-1 result in growth deficiencies in children. Administration ofIGF-1 can ameliorate the problem. It was found that IGF-1 levels innasal mucus are approximately 3-fold higher then IGF-1 levels in saliva(p<0.001). Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting intracellular adhesion molecule 1 (ICAM-1) or CD 54, atransmembrane protein that contains 2-9 extracellular Ig-like C-2 typedomains. ICAMs bind to LFA-1 integrins and mediate adhesion interactionwith cells of the immune system. It is differentially expressed onepithelial, endothelial and hematopoietic cells and may be expressed inbrain. Elevated levels of ICAM-1 are associated with carotidarteriosclerosis, cardiac ischemia, cardiac transplantation,subarachnoid hemorrhage (SAH), stroke, asthma, HIV, melanoma, andlymphoma. It was discovered that nasal mucus levels are approximately26% of plasma levels (p<0.001) with saliva at approximately 15% ofplasma levels. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting interferon alpha (IFN-α) a member of the Type I interferonfamily, that share a common receptor complex. IFN-α has both antiviraland immunomodulating activities on target cells. Increased levels ofIFN-α are associated with viral infections including hepatitis C. IFN-αis administered to treat viral infections including hepatitis C, andcancers such as bladder cancer, renal cell cancer, melanoma andchromamyelocyte leukemia. Nasal mucus monitoring represents a new methodto measure treatment levels of IFN-α in patients receiving IFN-αtreatment

It was discovered that nasal mucus levels of IFN-α are almost twice thatof plasma (p<0.01), while saliva was almost six-fold higher than plasma.Table 46. Patients with allergic diseases had increased levels of IFN-αcompared to controls.

Some embodiments of the invention include diagnosing a disease bydetecting interferon β (IFNβ), a member of the Type I interferon family.It shares a common cell surface receptor with IFNα and IFNγ. IFN-β is apotent antiviral substance. It can be used as an antiviral and antitumortreatment through nasal inhalation. IFN-β may also antagonize theeffects of IFN-γ and other proinflammatory cytokines, such as tumornecrosis factor and IL-1, and thereby down-regulate T-cell activity.

IFN-β is administered to treat hepatitis C, papilloma virus, multiplesclerosis, condylomatosis, lung fibrosis and melanoma. Nasal mucusmonitoring represents a new method to measure treatment levels of IFN-βin patients receiving IFN-β treatment. It was discovered that nasalmucus levels are approximately half of plasma levels, but the differencewas not statistically significant. Table 46. Nasal mucus levels areapproximately 15-fold greater than saliva levels. Patients with allergicdiseases had increased levels of IFN-β compared to controls, as did postinfluenza patients.

Some embodiments of the invention include diagnosing a disease bydetecting interferon-γ (IFN-γ), a type II interferon or immuneinterferon produced by T cells and natural killer (NK) cells. It sharesno significant homology with IFNβ or IFNα. It exists in mature form as anon-covalently linked homodimer Decreased levels of IFN-γ are associatedwith tuberculosis, chlamydia, atopic disease, and cervical cancer.Interferon-γ (INFγ) is the initial and primary inducer ofimmunoproteasomes during viral infection. It is a major cytokine in manyviral infections, and is known to alter the composition and function ofmany viral infections (4). IFN-γ induces the transcription andtranslation of the 3 immunoproteasome subunits β1i (LMP2), β2i (MECL-1)and β5i (LMP7) which replace their constitutive counterparts β1, β2 andβ5, respectively, during de novo assembly of proteosomes (5). INFγ playsan important role in the induction of immunoproteasomes as demonstratedin a murine model of fungal infection (5). Many viruses induce avigorous Type 1 INF response. IFN-γ can be found at the site of activeviral infection. It has been well studied as both an active and morechronic indication of viral disease. Decreased levels of IFN-γ areassociated with tuberculosis, chlamydia, atopic disease and cervicalcancer. Increased levels may be predictive of atopic eczema.

IFN-γ could not be detected in plasma, saliva or urine but it is foundonly in nasal mucus ranging from 0-333 pg/ml (85±18 pg/ml, Mean±SEM).Table 46. This finding in nasal mucus is independent of the presence ofblood or active infection in the nose. This finding suggests that thenasal cavity is an active portal of entry of viral particles into thebody and that IFN-γ in the nasal cavity reflects an important reactionto this entry. Its level can reflect endogenous susceptibility toinfection, resistance to infection and/or activity of infection. As suchits measurement may be an important step in recognizing how viruses ofany type alter human metabolism as related to immune system activity.Interestingly, these results with IFN-γ and other substances that areelevated in viral infections may provide useful surrogate markers todefine the extent and severity of a specific disease—e.g. levels of INFγin viral diseases such as hepatitis, HIV AIDS, etc. They may evensubstitute for measurements of active viral loads.

Some embodiments of the invention include diagnosing a disease bydetecting interferon-ω (IFN-ω), also known as leukocyte (II) interferon.IFN-ω is a monomeric glycoprotein distantly related to IFNα and β butunrelated to IFNγ. Its binding characteristics are similar to those ofother interferons. IFN-ω can be used to treat hepatitis B and C, otherviral infections and forms of cancer. It was discovered that nasal mucuslevels of IFN-ω are similar to plasma levels, while IFN-ω wasundetectable in urine. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting interferon receptor, comprising two subunits, IFNAR1 andIFNAR2. Nasal mucus levels were approximately 6-fold higher than salivalevels at 271±14 pg/ml versus 43.3 pg/ml. Interestingly, patients withallergic disease had nasal mucus levels of approximately 3.5-fold higherthan control levels, while post influenza patients had approximately4.6-folder higher level.

Some embodiments of the invention include diagnosing a disease bydetecting interleukin 1 alpha (IL-1), a cytokine produced by a varietyof cells in response to inflammation, infection and/or microbial toxins.Increased levels of IL-1 are associated with leukemia, solid tumors,Down's syndrome, Alzheimer's disease, HIV, meningococal shock, adultperiodontal disease, and ulcerative colitis. Increasing levels of IL-1may be predictive of premature labor. It was discovered that nasal mucuslevels are approximately 69-fold higher compared to saliva levels andover 1900-fold higher compared to plasma levels in normal individuals.Table 46. Additionally, nasal mucus levels are elevated in patients withhead injuries and decreased in patient with allergic disease. Someembodiments of the invention include diagnosing a disease by detectinginterleukin 1 receptor. Increased levels of interleukin 1 receptor areassociated with rheumatoid arthritis, osteoarthritis, silicosis,cerebral ischemia, stroke, traumatic brain injury, gastritis, septicshock, and abdominal aortic aneurysm. Increased levels may be predictivefor the development of peridontal disease and inflammatory skindiseases. It was discovered that nasal mucus levels of interleukin 1receptor are approximately 9% of plasma levels (p<0.001), while salivalevels were approximately 1.1% of plasma levels. Table 46. Increasedlevels of interleukin 1 receptor were noted in patients with headinjury.

Some embodiments of the invention include diagnosing a disease bydetecting interleukin 2 (IL-2), a T cell growth factor produced by Tcells following activation by mitogens or antigens. It has also beenshown to stimulate growth and inhibition of B cells, NK cells,lymphocyte-activated killer cells (LAK), monocytes/macrophages andoligodendrocytes. Increased IL-2 levels are associated with cancersincluding gastric cancer and melanoma and in diabetic nephropathy. Itwas discovered that IL-2 could only be detected in nasal mucus and notin the other bodily fluids. Table 46. Elevated levels of IL-2 were notedin post influenza patients and those with active allergies.

Some embodiments of the invention include diagnosing a disease bydetecting interleukin 2 receptor (IL-2 receptor). Increased levels ofIL-2 receptor are associated with myelodysplastics syndrome andleukemia. Increasing levels of IL-2 receptor may be predictive of thedevelopment of gastic cancer and metastatic disease and colon cancer andmetastatic disease. It was discovered that the level of IL-2 receptor innasal mucus was approximately 7% of plasma levels (p<0.001), whilesaliva levels were approximately 1.2% of plasma levels. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting interleukin 3 (IL-3), also known as mast cell growth factor.It is a pleotropic factor produced by activated T cells. IL-3 canstimulate proliferation and differentiation of pluripotent hematopoeiticstem cells as well as various lineage-committed progenitors. Increasedlevels of Il-3 are associated with myeloproliferative syndrome and acutemyelogenous leukemia. IL-3 was detectable in both nasal mucus andsaliva. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting interleukin 6 (IL-6), a multifunctional α helical cytokinethat plays important roles in host defense, acute phase reaction, immuneresponse and hematopoiesis. It is expressed by both normal andtransformed cells including T cells, B cells, monocytes/macrophages,fibroblasts, hepatocytes, keratinocytes, astrocytes, vascularendothelial cells and various tumor cells. Interleukin 6 (IL-6) binds toa high affinity receptor complex consisting of two membraneglycoproteins: an 80 KD component receptor that bind IL-6 with lowaffinity (IL6-R) and a signal-transducing component of 130 KD that doesnot bind IL-6 itself but is required for high-affinity binding of IL-6by the complex. Increased levels of IL-6 are associated withosteoporosis, rheumatoid arthritis, meningococcal shock, sepsis,systemic lupus erythemitosis, glomerulnephritis, prostate cancer andactive HIV. Increased levels of IL-6 may be predictive of futuremyocardial infarction, development of coronary disease, unstable angina,or preterm labor. It was discovered that IL-6 levels in nasal mucus areapproximately 17-fold greater than plasma levels (p<0.001). Table 46.Post influenza patients and those with allergic disease had about a3-fold higher level of IL-6 than controls.

Some embodiments of the invention include diagnosing a disease bydetecting interleukin 15 (IL-15), a cytokine with structural similarityto IL-2 that is secreted by mononuclear phagocytes following viralinfection. IL-15 induces cell proliferation of natural killer cells;cells of the innate immune system whose principal role is to killvirally infected cells. Increased levels of IL-15 are associated withrheumatoid arthritis, ulcerative colitis, inflammatory bowel disease,hepatitis C induced liver disease, hepatocellular carcinoma, cardiacdisease, fibromyalgia, lymphoproliferative disorder of granularlymphocytes (LDGL) and other chronic lymphoproliferative diseases, hairycell and B cell leukemia, and transplant rejection. It was discoveredthat IL-15 levels in nasal mucus are approximately 32-fold highercompared to plasma levels (p<0.001), while saliva levels were similar toplasma levels. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting interleukin 17 (IL-17), also known as IL-17A, a member of theIL-17 family that includes IL-17B, IL-17C, IL-17D, IL-17E (also calledIL-25), and IL-17F. All members of the IL-17 family have a similarprotein structure, yet they have no sequence similarity to any otherknown cytokines. Increased levels of IL-17 are associated withrheumatoid arthritis, MS, scleroderma, osteoarthritis, chronicobstructive pulmonary disease (COPD), and lung inflammation. Nasal mucusand saliva levels of IL-17 were similar, however, IL-17 was notdetectable in plasma. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting interleukin 18 (IL-18), also known asinterferon-gamma-inducing factor (IGIF) or IL-IF4, IL-18 shares biologicactivity with IL-12. It is synthesized as a propeptide that is cleavedto create a 19 KD monoglycosylated monomer. Increased levels of IL-18are associated with unstable angina, atherosclerosis, myocardialinfarction and other cardiovascular diseases, liver disease, diabetesmellitus type 1, systemic lupus erythematosus, biliary artesia,pancreatic necrosis, sarcoidosis, tuberculosis, and sepsis. It wasdiscovered that nasal mucus levels of IL-18 are similar to plasmalevels. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting keratinocyte growth factor (KGF), also known as FGF7, a memberof the fibroblast growth factor family. KGF is a paracrine-acting,epithelial mitogen produced by cells of mesenchymal origin and actsexclusively through a subset of FGF receptor isoforms (FGFR2b) expressedpredominantly by epithelial cells. The upregulation of KGF afterepithelial injury suggested it had an important role in tissue repair.Elevated levels of KGF are associated with breast cancer, inflammatorybowel disease and psoriasis. Decreased levels are associated withcongenital lung abnormalities, alopecia greata, and ulcerative colitis.It was discovered that nasal mucus levels of KGF are approximately5-fold higher than plasma levels (p<0.005), while KGF was undetectablein saliva. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting L selectin, also known as CD62L, a cell adhesion moleculefound on leukocytes. It belongs to the selectin family of proteins,which recognize sialylated carbohydrate groups. Elevated L selectinlevels are associated with diabetes mellitus, and colon cancer.Decreased levels are associated with myocardial injury and thoseundergoing acute inflammatory processes. Nasal mucus levels of Lselectin were not significantly different from plasma levels, while Lselectin was not detected in saliva. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting leptin, a 16 kDa protein hormone that plays a key role inregulating energy intake and energy expenditure, in part, by decreasingappetite and increasing metabolism. Administration of leptinintranasally is suggested to be the clinically beneficial manner toinhibit obesity. Leptin dosing can be monitored through nasal mucussampling.

Some embodiments of the invention include diagnosing a disease bydetecting leptin-receptor, a cellular receptor responsive to leptin withcharacteristics similar to insulin receptors.

Some embodiments of the invention include diagnosing a disease bydetecting leukemia inhibitory factor (LIF), a member of the interleukin6 family of cytokines. Functionally it has been implicated in manyphysiological processes including hematopoiesis, bone metabolism andinflammation. Some cell types known to express LIF include activated Tcells, monocytes, astrocytes, keratinocytes, regenerating skeletalmuscle, mast cells and fibroblasts. Elevated levels of LIF areassociated with rheumatoid arthritis. Decreased LIF levels areimplicated in deficiencies in placentation that result in earlyabortion, or pre-eclampsia and intrauterine growth restriction leadingto impaired fetal health, including intrauterine growth retardation,prematurity and maternal death. LIF was detected in nasal mucus, but notin plasma, urine or saliva. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting migration inhibitory factor (MIF), produced by activatedmacrophages where it sustains macrophage survival and function bysuppressing activation-induced, p53-dependent apoptosis. Increasedlevels are associated with glomerulonephritis, metastatic prostatecancer, rheumatoid arthritis. Increasing levels of MIF may be prognosticfor tumor growth including the development of lung cancer. Decreasedserum MIF concentrations during early gestation were found in pregnantwomen suffering from recurrent miscarriage of fetuses with normal fetalchromosome karyotype. Decreased levels are also associated with septicshock and respiratory distress syndrome. It was discovered that MIFlevels in nasal mucus are approximately 48-fold higher than in plasma(p<0.001), while MIF was not detectable in saliva or urine. Table 46.Some embodiments of the invention include diagnosing a disease bydetecting matrix metalloproteinase 1 (MMP-1), also known as interstitialcollagenase, a member of the MMP extracellular protease family. MMP-1plays a critical role in extracellular matrix remodeling under bothphysiological and pathological conditions. Additional substrates includecytokines, chemokines, growth factors, binding proteins, cell/celladhesion molecules and other proteinases. Increased levels of MMP-1 areassociated with chronic peptic ulcers, caroitid atherosclerosis, orallichen planus, oral carcinoma, aortic aneurysm, and esophageal cancer.Increasing levels of MMP-1 may be associated with poor prognosis ofcolon cancer patients. Decrease MMP-1 levels are associated withrheumatoid arthritis. Nasal mucus levels of MMP-1 were less than half ofplasma levels, but the difference was not statistically significant.Table 46. MMP-1 was not detectable in saliva and urine.

Some embodiments of the invention include diagnosing a disease bydetecting nerve growth factor (NGF), a three unit protein consisting ofα, β and γ subunits of which only the β subunit is physiologicallyactive. This is the 2.55 subunit which induces neurite outgrowth invarious tissues.

Some embodiments of the invention include diagnosing a disease bydetecting nitric oxide (NO), a signaling molecule produced from arginineby nitric oxide synthase. Endothelium cells release nitric oxide tosignal surrounding smooth muscle to relax, resulting in vasodilation andincreasing blood flow. NO is toxic to bacteria and other pathogens andis released by macrophages and neutrophils as part of the immuneresponse. Increased levels of NO are associated with asthma and breastcancer. Decreased levels are associated with atherosclerosis andhypertension. NO is also used as a treatment for respiratory distresssyndrome and pulmonary hypertension.

Some embodiments of the invention include prognosis a disease bydetecting the inactive N-terminal fragment from pro-brain natriureticpeptide (NT-proBNP) that is co-secreted with brain natriuretic peptide(BNP). NT-pro-BNP serum levels are predictive of mortality amongpatients with acute coronary syndrome and serve as a prognostic markerin patients with stable coronary heart disease and acute congestiveheart failure (CHF). NT-pro-BNP has been measured in serum samples ofpatients with acute and chronic coronary heart disease. NT-pro-BNPlevels were significantly lower in patients who survived than amongthose who died with acute or chronic disease [120 pg/ml for survival vs386 pg/ml for deaths (p<0.001)]. Patients with NT-pro-BNP levels in thehighest quartile were older, had a lower left ventricular ejectionfraction (LVEF), a lower creatinine clearance rate and were more likelyto have a history of myocardial infarction, clinically significantcoronary artery disease and diabetes than patients with NT-pro-BNPlevels in the lowest quartile. The hazard ratio for death for patientswith NT-pro-BNP levels in the highest quartile compared to those in thelowest quartile was 2.4 (p<0.001). This NT-pro-BNP level addedprognostic information beyond that provided by conventional risk factorsincluding patient age, sex, family history of ischemic heart disease,presence or absence of history of myocardial infarction, angina,hypertension, diabetes, chronic heart failure, creatinine clearancerate, body mass index, smoking status, plasma lipid levels, LVEF or thepresence or absence of clinically significant coronary artery disease orangiography. Thus NT-pro-BNP levels can be used, not only as an acutemarker, but also a marker of long-term mortality in patients with stablecoronary heart disease and provides prognostic information above andbeyond that provided by environmental cardiovascular risk functions anddegree of left ventricular systolic dysfunction. Now, it may be possibleto screen patients for NT-pro-BNP by sampling nasal mucus to provide asimple, rapid and low cost screening method to classify cardiac diseasepatients based on predicted mortality rate or prognosis and provide theappropriative therapeutic and support care based on the predictedmortality rate or prognosis.

Some embodiments of the invention include diagnosing a disease bydetecting P selectin, also known as CD62P, Granule Membrane Protein 140(GMP-140), or Platelet Activation-Dependent Granule to External MembraneProtein (PADGEM). P selectin is a cell adhesion molecule (CAM) found ingranules in endothelial cells (cells lining blood vessels) and activatedplatelets. P-selectin is involved in the initial recruitment ofleukocytes to injury sites during the initial inflammatory response.Elevated levels of P selectin are associated with atherosclerosis,hypertension, unstable angina, brain ischemia, diabetes mellitus, nasalpolyposis, and tumor metastasis. Decreasing levels may be predictive oflung injury. It was discovered that P selectin levels in nasal mucus areapproximately 5% of plasma levels (p<0.001). Table 46. P selectin wasnot detectable in saliva or urine.

The platelet derived growth factor (PDGF) family consists of PDGF-A, -B,-C and -D, which form either homo- or heterodimers. PDGF is theprincipal mitogen in serum for mesenchymal cells. Increased levels ofPDGF-AA are associated with meningioma, and renal tissue injury. PDGF-AAcan be used therapeutically for the treatment of chronic pressure ulcer,periodontal disease, non-healing wounds and pulmonary edema. Increasedlevels of PDGF-BB are associated with astrocytoma, and artheromaformation. Decreased PDGF-BB levels are associated with impaired woundhealing of diabetic patients. Nasal mucus levels of PDGF-AA wereslightly less than plasma levels, but the result was statisticallysignificant. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting placenta derived growth factor (PlGF), a member of thevascular endothelial growth factor (VEGF) family. PlGF exists in atleast 4 different isoforms as a result of alternative splicing. It wasfirst identified in human placenta and is expressed in various tissuesincluding blood vessels, human umbilical vein, endothelia, bone marrow,uterine NK cells, and keratinocytes. PlGF is upregulated under specificpathological conditions including wound healing, tumor function andpregnancy. It is an indicator of for predicting the development ofpreeclampsia during pregnancy and may be present in blood up to 10 weeksprior to onset of symptoms of this disorder. We have discovered, for thefirst time, the presence of PlGF in nasal mucus. In plasma, PlGF rangedfrom 0-79 ng/ml (19.5±7.6, Mean±SEM) whereas in nasal mucus PlGF rangedfrom 0-1402 ng/ml (350±90). Table 49. PlGF concentration in salivavaried from 0-65 pg/ml (16.6±6.2) and urine from 0-84 pg/ml (23.3±9.4).These results indicate PlGF in nasal mucus is 18 times higher than inplasma, 21 times than in saliva and 15 times higher than in urine. Thisindicates that PlGF is found in significantly higher concentrations innasal mucus than in other body fluids. Higher concentration coupled withthe ease of measurement and patient comfort and acceptance makes PlGF amore useful marker of PDF than is any other body fluid.

Furthermore, PlGF was found in nasal mucus in 94% of patients, but inplasma in only 65% of patients, in saliva in only 59% of patients and inurine in only 67% of patients. Thus, measurement of PlGF in nasal mucusis a significantly more reliable measurement than is plasma, saliva orurine (X², p<0.01).

These findings suggest that nasal mucus concentration of PlGF can bemeasured alone or in tandem with endoglin to predict the occurrence ofpreeclampsia prior to its onset of clinical symptomology. Through nasalmucus testing for biomarkers for the development of preeclampsia, it maybe possible to intervene before symptoms manifest and thereby ameliorateor avoid fetal growth restriction, appearance of the HELP syndrome(hemolysis, elevated liver enzymes and low platelets) and potentialfetal wastage associated with preeclampsia. It is critical to note thatlevels of endoglin and PGF were not elevated in patients who weredestined to have gestational hypertension or to remain normotensive butdeliver growth-restricted neonates.

Endoglin and PlGF can be measure individually or in combination usingnasal mucus as a diagnostic test for preeclampsia. With combinationtesting, separate diagnostic devices can be used or the two substancescan be tested simultaneously on the same device. Nasal mucus can besampled by the direct placement of a swab or similar sampling devicedirectly into one nasal naris. The collected nasal mucus can betransferred to a diagnostic device. Alternatively, nasal mucus can becollect using a swab or other collection means that is engineered into adiagnostic device. The device will have one or more substances thatreact with one or more biological substances present in nasal mucus. Inone embodiment, the reactive substances are antibodies to endoglin andPlGF. Upon application to the device or collection of nasal mucus withthe device, the biologic substances of interest will react directly withthe reactive substances and over the course of several minutes of thereaction, a colorimetric result will be readable. The device may furtherinclude a biologic substance reactive with a ubiquitously occurringnasal mucus substance so as to produce a positive control signaldemonstrating the correct functioning of the assay device. The simplecolormetric readout of the device will allow non-professional orlow-skilled health personnel in any environment, developed ornon-developed countries, to obtain rapid, specific quantitativemeasurements of both endoglin and PlGF without use of any externalequipment. This will allow the prediction of the occurrence ofpreeclampsia at least 10 weeks prior to the onset of symptoms of thispathology.

Some embodiments of the invention include diagnosing a disease bydetecting receptor for abdominal glycation end (RAGE) products, amulti-ligand transmembrane glycoprotein belonging to the immunoglobulinsuperfamily. RAGE ligands include advanced glycation end products (AGEs)amyloid-β and several members of the S-100 protein superfamily. Elevatedlevels of RAGE are associated with Alzheimer's disease, diabetesnephropathy, glomerulosclerosis, and macular degeneration. Increasinglevels of RAGE may be predictive for diabetes and peripheral vasculardisease. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting stem cell factor (SCF) also known as KIT ligand or Steelfactor. SCF is a cytokine that binds CD117 (c-Kit). SCF is an importantgrowth factor for the survival, proliferation, and differentiation ofhematopoietic stem cells and other hematopoietic progenitor cells.Increased levels of SCF are associated with breast cancer, testicularcancer, gynecological cancers, leukemia, and cutaneous mastocytosis. Itwas discovered that nasal mucus levels of SCF were approximately 4% ofplasma levels (p<0.001). Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting substance P, an 11 amino acid peptide that belongs to thetachykinin neuropeptide family. It is synthesized as a large protein andthe enzymatically converted to its active form. Substance P is widelydistributed in the central and peripheral systems where it acts as aneurotransmitter. In the peripheral nervous system it is localized toprimary sensory neurons and neurons intrinsic to the gastrointestinaltract. Increased levels of Substance P are associated with fibromyalgia,pancreatitis, Raynaud's phenomena, and Rheumatoid arthritis. Decreasedlevels are associated with psoriasis. It was discovered that nasal mucuslevels of substance P were similar to plasma levels and about twice thelevel found in saliva. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting tissue inhibitor of metalloproteinase (TIMP-1), one member ofa group of five TIMPs, each of which is capable of inhibiting almostevery member of the matrix metalloproteinases. Increased levels ofTIMP-1 are associated with breast, colon, and gastric cancer. Increasinglevels of TIMP-1 are associated with poor prognosis in patients withbreast, colon or gastric cancer. It was discovered that nasal mucuslevels of TIMP-1 were approximately 10-fold higher than plasma levels(p<0.001) and 20-fold higher than saliva levels (p<0.001). Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting transforming growth factor alpha (TGFα), a member of the EGFfamily of cytokines. TGFα plays a role in cell-cell adhesion and injuxtacrine stimulation of adjacent cells. Its expression is widespreadin tumors and transformed cells. It is expressed in normal tissuesduring embryogenesis and in adult tissues including pituitary, brain,keratinocytes and macrophages. Increased levels of TGF-α are associatedwith various cancers including squamous cell cancer of the head andneck, breast cancer, esophageal cancer, colon cancer and liver cancer.It was discovered that TGF-α was detectable in nasal mucus, but wasundetectable in plasma, saliva or urine. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting transforming growth factor-β (TGF-β). TGF-β acts as a potentgrowth inhibitor for most types of cells. Decreased levels of TGF-βassociated with the formation of cleft palate and abnormal neonatal lungdevelopment. Increased levels may be predictive for the development ofrenal disease including renal fibrosis. It was discovered that nasalmucus levels of TGF-β were over twice plasma levels (p<0.05) and alsoover twice saliva levels. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting triggering receptor expressed on myeloid cells (TREM-1), acell surface molecule on neutrophils and monocytes/macrophagesimplicated in the amplification of inflammatory responses by enhancingdegranulation and secretion of proinflammatory mediators. Increasedlevels of TREM-1 are associated with septic shock, sepsis, acutepancreatitis, and pneumonia.

Some embodiments of the invention include diagnosing a disease bydetecting tumor necrosis factor (TNF), also known as cachexin orcachectin and formally known as tumor necrosis factor-alpha) is acytokine involved in systemic inflammation and is a member of a group ofcytokines that stimulate the acute phase reaction. Increased levels ofTNF are associated with psoriasis, rheumatoid arthritis, Crohn'sdisease, glomerulonephritis, Parkinson's disease, head injury includingischemic brain injury, and dementia. Increased levels of TNF may bepredictive for the development of insulin resistance. It was discoveredthat nasal mucus levels of TNF were approximately 65% of plasma levelsand over 5-fold higher than saliva levels. Table 46. TNF levels in nasalmucus are elevated almost 6-fold in post influenza patients almost7-fold in post surgery patients.

Some embodiments of the invention include diagnosing a disease bydetecting tumor necrosis factor beta (TNFβ), also known as lymphotoxinalpha (LTα). TNFβ binds to cell surface receptors and produces a vastrange of effects. Increased levels of TNFβ are associated with B celllymphomas and multiple sclerosis. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting tumor necrosis factor receptor 1 (TNF-R1) is also known asP55/P60 TNFR. TNF-R1 is a high affinity receptor for TNFα and β. It isinvolved in the mediation of multiple aspects of inflammatory immuneresponse. TNF-R1 forms homodimers on oligomers upon ligand binding.Monomers have an extracellular domain which includes four cysteine richrepeats. Increased levels of TNF-R1 are associated with obesity,rheumatoid arthritis, and chronic inflammatory diseases. It wasdiscovered that nasal mucus levels of TNF-R1 are approximately 60% ofplasma levels (p<0.01), but are almost 8-fold higher than saliva levels.Table 46. Elevated nasal mucus levels were noted in patients with headinjury or those that are post influenza.

Some embodiments of the invention include diagnosing a disease bydetecting tumor necrosis factor receptor 2 (TNF-R2) is also known asP75/P80 TNFR. TNF-R2 is a receptor protein in the same family as TNFR1with some similar characteristics to TNFR1. Increased levels of TNF-R2are associated with idiopathic pulmonary fibrosis and may be predictiveof insulin resistance. Decreased levels may be predictive of liverdisease. It was discovered that TNF-R2 levels in nasal mucus areapproximately 50% of plasma levels, but over 9-fold higher than salivalevels. Table 46. Elevated levels of TNF-R2 were discovered in patientswith allergic diseases or oral burns.

Some embodiments of the invention include diagnosing a disease bydetecting TNF-related apoptosis-inducing ligand (TRAIL) is also known asapo-2 ligand and TNFSF-10. It is a type of Type II transmembrane proteinwith a carboxy terminal extracellular domain that exhibits homology toother TNF superfamily members. It is the most homologous to FAS-ligandsharing 28% amino acid identity. It reflects an end product of cellularapoptosis. Patients with allergic diseases and those post surgery hadelevated nasal mucus levels.

Some embodiments of the invention include diagnosing a disease bydetecting vascular cell adhesion molecule (VCAM1), a member of theimmunoglobulin superfamily. It is a cell surface protein expressed byactivated endothelial cells and certain leukocytes such as macrophages.Expression of VCAM1 is induced by IL-1β, IL-4, TNFα and IFNγ. It bindsto leukocyte integrins; VLA-4 and integrin α2 β7. Elevated levels ofVCAM1 may be predictive of early atherosclerosis and the development oflupus nephritis. Nasal mucus levels of VCAM1 were approximately 11% ofplasma levels and similar to saliva levels. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting members of the vascular endothelial growth factor (VEGF),family. This family is related to PlGF and modulates variousphysiological and pathological processes including vasculogenesis,angiogenesis and lymphangiogenesis.

One VEGF family member is VEGF-C, also known as Flt4 ligand is animportant and specific regulatory factor for lymphatic endothelialproliferation and lymphangiogenesis. Increased levels are associatedwith prostate cancer. Increased or increasing levels of VEGF-C may bepredictive of breast, gastric, and head and neck cancer metastasis. Itmay also be predictive for the development of colon cancer. It wasdiscovered that VEGF-C levels in nasal mucus are approximately 10-foldhigher than plasma levels (p<0.001) and approximately 9-fold higher thansaliva levels. Table 46.

Another VEGF family member is VEGF-D, an important and specificregulatory factor for lymphatic endothelial proliferation andlymphangiogenesis. Increased VEGF-D serum levels are significantlyelevated in patients with angiosarcoma. Increased or increasing levelsof VEGF-D may be predictive of tumor metastasis in the lymphatic systemand of ovarian cancer metastasis. It was discovered that nasal mucuslevels of VEGF-D are approximately 6% of plasma levels (p<0.001), butover 4-fold higher than saliva levels. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting vascular endothelial growth factor receptor 1 (VEGFR1). VEGFR1binds VEGF-A and is thought to modulate VEGFR-2 signaling. VEGFR1 is amember of the class III superfamily of receptor tyrosine kinases (RTKs).All receptors contain 7 immunoglobulin-like repeats in theirextracellular domains and kinase insert domains in their intracellularregion. They regulate VEGF mediated vasculogenesis, angiogenesis andlymphangiogenesis. They also mediate neurotrophic activity and regulatehematopoietic development. Increased or increasing levels of VEGFR1 maybe prognostic for endometrial cancer. It was discovered that nasal mucuslevels of VEGF-R1 are approximately 4-fold higher than plasma levels and3-fold higher than saliva levels. Table 46.

Some embodiments of the invention include diagnosing a disease bydetecting vascular endothelial growth factor receptor 2 (VEGFR2).Increased or increasing levels of VEGFR2 may be prognostic for oxygeninduced retinopathy and vascular retinopathy. It was discovered thatnasal mucus levels of VEGFR2 are approximately 7% of plasma levels(p<0.001), but 3.5-fold higher than saliva levels. Table 46.

Trace elements can also be measured in nasal mucus. Wilson's disease,also known as hepatolenticular degeneration is an example of a diseasethat can be diagnosed and/or prognosed through nasal mucus sampling.Table 47. Wilson's disease is an autosomal recessive disorder whereincopper accumulates in tissue in resulting in liver disease andneurological symptoms including taste and smell deficits. Wilson'sdisease is currently diagnosed by measuring ceruloplasmin in blood. Over80% of Wilson disease patients have suppressed levels of ceruloplasmin,typically about 30% of normal. More accurate measurements can beobtained by assaying urine, blood or a liver biopsy sample for copper.Of particular importance is the observation that increased tissue levelsof copper is predictive of the later development of Wilson's disease in95-100% of the individuals tested. Additionally, abnormal copper levelscan be observed one to ten years before symptoms appear allowingtreatment with chelating agents to reduce tissue levels of copper andprevent future build up along with instructions to follow a low-copperdiet.

It was discovered that patients with Wilson's disease have abnormallylow levels of copper in their nasal mucus, with an approximately 50-foldreduction compared to controls, 2±1 (mg/l) and 99±8 (mg/l),respectively. Table 50.

Patients with other taste and smell deficits appear to have increasedcopper levels in their nasal mucus, saliva and urine compared tocontrol. Table 50. Nasal mucus sampling for copper represents a simple,non-invasive technique that can be used to screen, or prognose for thedevelopment of Wilson's disease in addition to diagnosing the disease.

Zinc is another element that is associated with taste and smell loss. Inpatients with a taste and smell deficit that is not attributable toWilson's disease, nasal mucus contain over twice the level of zinc at236±37 (mg/l) compared to controls at 96±10 (mg/l). Table 51. Again,nasal mucus represents a simple, non-invasive technique to ascertaintrace element levels in a patient.

Measurement Techniques

We have measured these substances in blood, urine, saliva and nasalmucus using a 96 plate colorimetric ELISA procedure. This procedure,while very sensitive, is complex, time consuming and requires specialequipment for performance of the procedure. We plan to reduce thiscomplex technique to a point-of-care (POC) nasal swab test. For thistechnique we intend to insert a nasal swab directly into the nose, theswab coated as noted below such that the presence of nasal mucus in thenose will provide the antigen for the POC test. We would also develop atest such that secreted nasal mucus could be deposited directly onto theswab and the colorimetric POC test performed outside of the bodydirectly on the swab. By use of this technique we will measure theseprognostic, diagnostic substances independent of blood in a safe, rapidand powerful manner to screen patient with these potential disordersworldwide and thereby, with the initiation of appropriate therapy,utilize these diagnostic techniques to ward off onset of symptoms ofthese diseases prior to the onset of the full-blown disease pathology.

We have been involved in the development of a simplified ELISA procedurein which the nasal swab contains all the necessary colorimetrics toperform an ELISA procedure. There are several procedures we have beendeveloping to perform this test. One such procedure involves alateral-flow immunoassay rapid strip test (6). This technique has beenused previously in several diagnostic test devices and can be adapted touse quantum dots, SNP detection, nucleic acid detection, hexapet andother novel labeling techniques. This embodies a chromatographic devicewhich employs nanoparticles of antibody coated with materials that bindto the analyte to be measured (in nasal mucus) within the specifiedenvironment of the nasal swab. The analyte-nanoparticle complex flowslaterally through a series of overlapping membranes until it is capturedon the antibody line achieving a specific antigen-antibody complex. Avisual result, eliciting a color is achieved within a few minutes. Acontrol line is always available to define specificity. Even anunskilled operator can readily interpret the test results on asemi-quantitative basis without any need for complex or expensiveequipment. We plan to adapt this general technique to a swab (plastic orsome similar structure) which we will place into the nose to read thereaction directly on the swab or to take expelled nasal mucus and applyit directly onto the swab (as noted above) to read the reactionindependent of the nose per-se. These adaptations involve noveltechnology which is part of this new art and will allow rapid and simplesubstance identification using adaptations of the current availabledevices. By use of this new technology which we are proposing in thisapplication we intend to demonstrate that many substances using presentday technology are present in nasal mucus at levels below the thresholdfor present day determination. We intend to demonstrate that this newtechnology we propose will allow identification of these substances innasal mucus by use of a combination of amplification methods. One methodfor accomplishing this is to use a combination which will include bothimmunoassay and PCR methodologies which can detect substances in nasalmucus at a sensitivity approximately that of detection of nucleic acids.This technique is embodied in a modification of a prior technique calledimmuno-PCR (IPCR) which was first described in 1992 (7). By thismodification we project that we can detect all necessary diagnosticsubstances in nasal mucus by adsorbing the required substance (as theantigen) onto microtiter plate wells through contact with a series ofsubstances-antibody, followed by protein A avidin chimera and then bybiotinylated plasmid DNA. Detection will be amplified by eliminating thebackground signal which previously limited sensitivity. The details ofthis procedure and others that may also be used will be subjects ofother applications since the purpose of this application is to presentmainly the use of nasal mucus and the substances contained therein asprognostic, diagnostic indicators for the diagnosis of human physiology,pathology and disease.

Examples of Diseases

Without limiting the scope of the present invention, the examples ofsome of the diseases which can be diagnosed by detecting the biologicalsubstance, is provided herein. However, these examples are not intendedto limit the scope of the invention. The disease as provided hereininclude, infections, hematological disorders, oncological disorders,endocronological disorders, metabolic disorders, immunologicaldisorders, neurological disorders, vascular disorders, mast celldisorders, psychiatric disorders, neoplastic disorders, nutritionaldisorders, post irradiation disorders, and changes in the trace metalmetabolism.

Infectious diseases include acute and chronic parasitic and/orinfectious diseases from bacterial, viral or fungal sources, but are notlimited to, single or multiple cutaneous lesions, mucosal disease,chagas' disease, toxoplasmosis, leishmaniasis, trypanosomiasis,shistosomiasis, cryptosporidiosis, mycobacterium avium infections,leprosy, dengue, yellow fever, inner ear infections, urinary tractinfections, bacterial endocarditis, osteomyelitis, h. pylori associatedulcers, antibiotic associated colitis, sexually transmitted diseases,malaria, rheumatoid arthritis, inflammatory bowel disease, interstitialcystitis, fibromyalgia, autonomic nervous dysfunction, pyodermagangrenosum, chronic fatigue, chronic fatigue syndrome, sepsis syndrome,cachexia, circulatory collapse and shock resulting from acute or chronicbacterial infection, AIDS (including symptoms of cachexia, autoimmunedisorders, AIDS dementia complex and infections), wegnersgranulomatosis, aneurysms, hemorrhoids, sarcoidosis, chronicinflammatory bowel disease, Crohn's disease, vascular inflammatorypathologies, such as, but not limited to, disseminated intravascularcoagulation, atherosclerosis, and Kawasaki's pathology, inflammatorydiseases such as coronary artery disease, hypertension, stroke, asthma,chronic hepatitis, multiple sclerosis, peripheral neuropathy, chronicvascular headaches (including migraines, cluster headaches, and tensionheadaches), demyelinating diseases, such as multiple sclerosis and acutetransverse myelitis, extrapyramidal and cerebellar disorders, such aslesions of the corticospinal system, disorders of the basal ganglia orcerebellar disorders, hyperkinetic movement disorders such asHuntington's chorea and senile chorea, drug-induced movement disorders,such as those induced by drugs which block CNS dopamine receptors,hypokinetic movement disorders, such as Parkinson's disease, progressivesupranucleo palsy, cerebellar and spinocerebellar disorders, such asastructural lesions of the cerebellum, spinocerebellar degenerations(spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations,multiple systems degenerations (mencel, Dejerine-Thomas, Shi-Drager, andMachado Joseph)), systemic disorders (Refsum's disease,abetalipoprotemia, ataxia, telangiectasia, and mitochondrialmulti-system disorder), disorders of the motor unit, such as neurogenicmuscular atrophies (anterior horn cell degeneration, such as amyotrophiclateral sclerosis, infantile spinal muscular atrophy and juvenile spinalmuscular atrophy), Alzheimer's disease, Down's Syndrome in middle age,diffuse Lewy body disease, senile dementia of lewy body type,Wernicke-Korsakoff syndrome, chronic alcoholism, Creutzfeldt-Jakobdisease, subacute sclerosing panencephalitis, Hallerrorden-Spatzdisease, and Dementia pugilistica, dermatophytosis (e.g.,trichophytosis, etc), pityriasis versicolor, candidiasis,cryptococcosis, geotrichosis, trichosporosis, aspergillosis,penicilliosis, fusariosis, zygomycosis, sporotrichosis, chromomycosis,coccidioidomycosis, histoplasmosis, blastomycosis,paracoccidioidomycosis, pseudallescheriosis, mycetoma, mycotickeratitis, otomycosis, and pneumocystosis.

Ocular neovascularization, psoriasis, duodenal ulcers etc can also betreated when demonstrated by the diagnostic procedures described herein.Similarly, other diseases (their biological substances in parenthesis),include, but not limited to, neutropenia, gout, dwarfism or congenitalshort stature (growth hormone releasing hormone (GHRH)); congestiveheart disease (atrial natriuretic peptide or factor (ANP or ANF));osteoporosis (parathyroid hormone (PTH)); Paget's disease (calcitonin);accromegally, insulin sparing effects, treatment of long termcomplications of diabetes and treatment of various endocrine secretingtumors (somatostatin); Addison's Disease and Cushing's Syndrome,shipping fever (bovine respiratory syndrome) or ulcers, andstress-induced immunosuppression (corticotrophin releasing factor(CRF)); contraception, fertility control, suppression or interruption ofheat, treatment of ovarian cysts, precocious puberty, prostatichyperplasia and tumors, gynecologic diseases, and termination ofpregnancy (luteinizing hormone-releasing hormone (LHRH)); aplasticanemia, paroxysmal nocturnal hemoglobinurea, chronic myelocyticleukemia, polycythemia vera, essential thrombocythemia, myelofibrosis,myelodysplastic syndrome and acute leukemia; and hematological diseasessuch as megaloblastic anemia, AIDS, multiple myeloma, metastatic cancerof the bone marrow, and drug-induced myelosuppression (hematopoieticstem cell growth factor (SCGF)); body weight disorders, includingobesity, cachexia, and anorexia, and diabetes, neoplasms, andhyperamylinemia (agouti-related protein); impairment of functions,increased ceramide formation, which triggers nitric oxide-mediatedlipotoxicity and lipoapoptosis, obesity and hyperphagia (leptin);hypolipidimia, coronary heart disease, Niemann Pick Disease, Gaucher'sdisease, Batten's syndrome, Farber's lipogranulomatosis, Krabbe'sdisease, metachromic leukodystrophy, Tay-Sach's disease, GM1gangliosidoses, Fabry's disease, cystinosis, aspartylglycosaminuria(lipid profile which includes triglycerides, LDL-cholesterol andHDL-cholesterol), and generalized vascular disease, chronichyperglycemia, obesity, hypertension, atherosclerosis and heart disease,carbohydrate deficient glycoprotein syndrome type 1a, glycogenoses, andgalactosemia (carbohydrates).

Detection of the concentration of the caspases in the nasal secretioncan provide diagnosing a disorder or selection of therapeutic strategiesinvolving, e.g., inappropriate apoptosis and/or excessive cellproliferation, such as an inflammatory disease, a neurodegenerativedisease, cancer, a cardiovascular disease and, any disorder or diseasecharacterized by a gradual and prolonged development of apoptosis.Apoptosis functions in maintaining normal tissue homeostasis in avariety of physiological processes including embryonic development,immune cell regulation, normal cellular turnover and programmed celldeath of cancer cells. Thus, the dysfunction or loss of regulatedapoptosis can lead to a variety of pathological disease states. Forexample, the loss of apoptosis can lead to the pathological accumulationof self-reactive lymphocytes such as occurs in many autoimmune diseases.Inappropriate loss of apoptosis can also lead to the accumulation ofvirally infected cells and of hyperproliferative cells such asneoplastic or tumor cells. Inappropriate activation of apoptosis cancontribute to a variety of diseases such as AIDS, neurodegenerativediseases and ischemic injury.

Dysregulation of apoptosis has been implicated in numerous diseases suchas neurodegenerative disorders including Alzheimer's disease,Parkinson's disease, Huntington's disease, and amyotrophic lateralsclerosis (ALS), cerebellar degeneration, stroke, traumatic braininjury, CNS ischemic reperfusion injury including neonatalhypoxic-ischemic brain injury or myocardial ischemic-reperfusion injury,injury caused by hypoxia, cardiovascular diseases (e.g., myocardialinfarction), especially those which are associated with apoptosis ofendothelial cells, degenerative liver disease, multiple sclerosis,rheumatoid arthritis, hematological disorders including lymphoma,leukemia, aplastic anemia, and myelodysplastic syndrome, osteoporosis,polycystic kidney disease, AIDS, myelodysplastic syndromes, aplasticanemia and baldness. Diseases of the eye include glaucoma, retinitispigmentosa and macular degeneration.

Inflammatory disease states include systemic inflammatory conditions andconditions associated locally with migration and attraction ofmonocytes, leukocytes and/or neutrophils. Inflammation may result frominfection with pathogenic organisms (including gram-positive bacteria,gram-negative bacteria, viruses, fungi, and parasites such as protozoaand helminths), transplant rejection (including rejection of solidorgans such as kidney, liver, heart, lung or cornea, as well asrejection of bone marrow transplants including graft-versus-host disease(GVHD)), or from localized chronic or acute autoimmune or allergicreactions. Autoimmune diseases include acute glomerulonephritis;rheumatoid or reactive arthritis; chronic glomerulonephritis;inflammatory bowel diseases such as Crohn's disease, ulcerative colitisand necrotizing enterocolitis; granulocyte transfusion associatedsyndromes; inflammatory dermatoses such as contact dermatitis, atopicdermatitis, psoriasis; systemic lupus erythematosus (SLE), autoimmunethyroiditis, multiple sclerosis, and some forms of diabetes, or anyother autoimmune state where attack by the subject's own immune systemresults in pathologic tissue destruction. Allergic reactions includeallergic asthma, chronic bronchitis, acute and delayed hypersensitivity.Systemic inflammatory disease states include inflammation associatedwith trauma, burns, reperfusion following ischemic events (e.g.thrombotic events in heart, brain, intestines or peripheral vasculature,including myocardial infarction and stroke), sepsis, ARDS or multipleorgan dysfunction syndrome. Inflammatory cell recruitment also occurs inatherosclerotic plaques.

Examples of pathological conditions resulting from increased cellsurvival include cancers such as lymphomas, carcinomas andhormone-dependent tumors (e.g., breast, prostate or ovarian cancer).Abnormal cellular proliferation conditions or cancers that may betreated in either adults or children include solid phasetumors/malignancies, locally advanced tumors, human soft tissuesarcomas, metastatic cancer, including lymphatic metastases, blood cellmalignancies including multiple myeloma, acute and chronic leukemias,and lymphomas, head and neck cancers including mouth cancer, larynxcancer and thyroid cancer, lung cancers including small cell carcinomaand non-small cell cancers, breast cancers including small cellcarcinoma and ductal carcinoma, gastrointestinal cancers includingesophageal cancer, stomach cancer, colon cancer, colorectal cancer andpolyps associated with colorectal neoplasia, pancreatic cancers, livercancer, urologic cancers including bladder cancer and prostate cancer,malignancies of the female genital tract including ovarian carcinoma,uterine (including endometrial) cancers, and solid tumor in the ovarianfollicle, kidney cancers including renal cell carcinoma, brain cancersincluding intrinsic brain tumors, neuroblastoma, astrocytic braintumors, gliomas, metastatic tumor cell invasion in the central nervoussystem, bone cancers including osteomas, skin cancers includingmalignant melanoma, tumor progression of human skin keratinocytes,squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma andKarposi's sarcoma.

Viral infections that may be detected include infections caused byherpesviruses (including CMV, HSV-1, HSV-2, VZV, EBV, HHV-6, HHV-7 andHHV-8), paramyxoviruses (including parainfluenza, mumps, measles, andrespiratory syncytial virus (RSV)), picornaviruses (includingenteroviruses and rhinoviruses), togaviruses, coronaviruses,arenaviruses, bunyaviruses, rhabdoviruses, orthomyxoviruses (includinginfluenza A, B and C viruses), reoviruses (including reoviruses,rotaviruses and orbiviruses), parvoviruses, adenoviruses, hepatitisviruses (including A, B, C, D and E) and retroviruses (including HTLVand HIV). Treatments of both acute and chronic infection arecontemplated.

Adenylyl cyclases are a family of enzymes that catalyze the formation ofAdenosine-3′:5-cyclic monophosphate (cAMP) fromadenosine-5′-triphosphate (5′ATP), mediate the physiological effects ofnumerous hormones and neurotransmitters, and belong to a super family ofmembrane-bound transporters and channel proteins. Adenosine-3′:5′-cyclicmonophosphate (cAMP) is the second messenger involved in signaltransduction for numerous neurotransmitters and hormones, and thus mayhave an impact upon some of the mediators for smooth muscle cells (SMC)proliferation and migration. Many hormones and other substances mayactivate cAMP and may activate the subsequent signaling cascades viatheir indirect influence on adenylyl cyclase. cAMP is a growth factorfor neurite growth and is involved in development in tissue culture ofsympathetic ganglion cells similar to the action of NGF. Adenylylcyclase is a growth factor which acts on stem cells in taste buds andolfactory epithelium to induce growth and development of all cell typesin taste buds and olfactory epithelium. cGMP, guanosine 3′, 5″-cyclicmonophosphate is formed by the action of guanylyl cyclase on GTP. cGMPis present at levels typically lower than cAMP in most tissues.Hormones, such as insulin and oxytocin as well as other substancesincluding acetylcholine, serotonin and histamine may increase cGMPlevels. Stimulators of cGMP may include vasodilators and peptides thatrelax smooth muscle.

Adenylyl cyclases also play a role in the disease progression ofCongestive heart failure (CHF). CHF is defined as an abnormal heartfunction resulting in an inadequate cardiac output for metabolic needs.Heart failure is usually not recognized until a more advanced stage ofheart failure which is referred to as congestive heart failure. Onphysical examination, patients with CHF tend to have elevations in heartand respiratory rates, rates (an indication of fluid in the lungs),edema, jugular venous distension, and, in general, enlarged hearts. Themost common cause of CHF is atherosclerosis which causes blockages inthe blood vessels (coronary arteries) that provide blood flow to theheart muscle. Ultimately, such blockages may cause myocardial infarction(death of heart muscle) with subsequent decline in heart function andresultant heart failure.

Fibroproliferative vasculopathy includes restenosis following coronarybypass surgery and PTCA (percutaneous transluminal coronaryangioplasty), allograft arteriosclerosis in chronic allograft rejection,diabetic angiopathy and all forms of common arteriosclerosis. Vascularintimal dysplasia and remodeling are characteristic features of reinjuryfollowing balloon angioplasty, coronary bypass surgery and in chronicallograft rejection. An initial response to vascular injury isinflammatory and involves attraction of lymphocytes, macrophages andthrombocytes to the site of injury and secretion of cytokines,eicosanoids and growth factors. Under the influence of growth factorsand cytokines, smooth muscle cells (SMC) may proliferate and migratefrom the media to the intima and contribute to intimal hyperplasia andstenosis. cAMP has an impact upon some of the key mediators for SMCproliferation and migration.

Glucose is irreversibly oxidized within the cells to produce water andcarbon dioxide. In the presence of a catalyst, especially a carbonicanhydrase enzyme (of which several forms exist, of which the formpresent depends upon the type of tissue cells present), the water andcarbon dioxide may reversibly produce a hydrogen ion and a bicarbonateion. Hydrogen ion produced by carbonic anhydrase enzymes can be actedupon by cytochrome system, which can then be utilized as the energysource of the ion pump that maintains the integrity of the cell membranecomprising and enclosing each cell. It can also be a source of thebrain's electric current. Disruption of the process may causedepolarization of the cell wall membrane, hence sodium (Na), water, andother chemicals can enter the cell in uncontrolled amounts and potassium(K) can exit uncontrollably, leading to the death and destruction of theinvolved cells followed by cellular edema. As this edema progresses, thecell dies. Along with the progressive and gradual death of cells,gliosis may follow resulting in the aging in the brain. The deficiencyof carbonic anhydrase can cause conditions of aging associated with adecreased presence of cell-specific carbonic anhydrase enzymes in thebrain, such as chronic neurodegenerative conditions including dementiasuch as Alzheimer's disease, or showing other forms of dementia orneurodegenerative diseases.

Methods of Treatment

The substances secreted into saliva and nasal mucus act on local oraland nasal tissues, respectively, to induce physiological effects. Thereare several effects of gland secretion at distant sites: (1)endocrine-secretions from a gland and subsequent action at a distantsite, the secretion carried in blood to the distant site; (2)paracrine-secreted substances act at a distant site within the localreach of the fluid; (3) exocrine-secretions from a gland which havedirect local effects, e.g., β-cells in the pancreas which act directlyto secrete insulin in response to local changes in blood glucose. Thisis a one directional effect, a secretion from the gland, into thebiological fluid, acting at a distant but local site.

There are feedback mechanisms such that whatever effects the glandsecretion had on its receptor, the receptor also interacted with thesite of secretion. For example, increased glucose induces increasedsecretion of insulin but as insulin secretion increases, insulinreceptor number in liver and pancreas change in response to theincreased insulin secretion. This feedback concept can also beexemplified by brain secretion of peptide hormones which acted as masterfeedback mechanisms to control peripheral hormone secretion. Thus, thereare interactions between brain, gland and a receptor with theinteractions proceeding in both directions. For example, TRH secretedfrom the brain hypothalamus stimulates pituitary TSH which acts tostimulate thyroid T₃ and T₄ which can act back on both pituitary andbrain in the form of both long (to brain) and short (to pituitary)feedback loops.

Henkin, R. I., Olfaction and Taste XI, (Kurihara, K., Suzuki, N., Ogawa,H., Eds.), Springer Verlag, 1994, pp. 568-573, incorporated herein byreference in its entirety, described the concept involving saliva andnasal mucus secretions related to taste and smell function (FIGS. 11 and12). These results suggest that tastants and odorants affect brainfunction and vice versa. Since saliva and nasal mucus are the criticalfactors in maintaining the taste and smell systems, respectively, it isunderstandable that substances in these fluids also affect brainfunction and vice versa. Therefore, nasal administration of substancescan affect brain function and thereby affect various physiological andpathological problems. For example, nasal administration of leptin (tocontrol obesity), agouti-related protein (to increase appetite inanorexic patients), glucose, albumin, insulin (to treat diabetes),hormones (hormonal disorders), etc.

These effects may act through the large arteriovenous plexus of bloodvessels in the nose such that absorption of the substances may beenhanced by direct contact and absorption through these exposed vessels.FIGS. 11 and 12 reflect a feedback mechanism with effects acting fromnose to brain and from brain to nose, as in both a short and long loopfeedback system.

Treatment with Drug

Theophylline treatment restores smell function in some patients withhyposmia (loss of smell). Theophylline is a phosphodiesterase (PDE)inhibitor; it restores smell function through PDE inhibition therebyincreasing cAMP, a growth factor which stimulates maturation ofolfactory epithelial stem cells, cells whose functions are inhibitedamong patients with hyposmia. Theophylline may also restore smellfunction through other mechanisms. One such mechanism may operatethrough inhibition of excessive apoptosis, a normal process which, ifexcessively increased, can become pathological and impair cellularanatomy of the olfactory epithelium and cause hyposmia.

Table 16 illustrates detection and measurement of TRAIL in nasal mucusin patients with hyposmia before and after treatment with theophyllineat various doses. Data indicated that treatment with theophylline whichreturned smell function to normal in a dose-dependent manner wasassociated with a dose-dependent decrease in TRAIL. These data indicatethat treatment with a drug demonstrated a dose dependent decrease inTRAIL which indicates a decrease in the abnormal apoptotic processes.These data also indicate both a biochemical and functional improvementin smell function by treatment with theophylline. Without limiting thescope of the present invention, other drugs are also considered with inthe scope of the present invention for the treatment of variousdiseases. This is one of the example of the multiple examples of drugsto treat disease in which changes of various substances found in nasalmucus reflect biochemical normalization and functional improvement inthe disease process.

Table 33 in the examples illustrates NO in nasal mucus in patientstreated with theophylline in various doses before and after drugtreatment. NO levels in nasal mucus changed following the treatment ofpatients with smell loss. Results show treatment of patients with gradedincreasing doses of theophylline and measurement of both smell functionand NO in nasal mucus in patients with hyposmia. Results indicated thatprior to the treatment levels of NO in nasal mucus were lower than innormal subjects. After treatment with theophylline in graded doses therewere increases in nasal mucus NO associated with graded increases insmell function. These data demonstrate that treatment with drugs thatincrease smell function to or toward normal, returns smell function tonormal. These results demonstrate the measurements of various substancesin nasal mucus as an index of both human physiology and pathology ofvarious diseases. Its continual measurement during treatment of thedisorders helps in monitoring efficacy of therapy. The detection of NOin nasal mucus provides a non invasive method of diagnosing variousdiseases related to human physiology and pathology. The methods of thepresent invention include treatment of diseases by modulating theconcentrations of NO by use of drugs or agents. The method of treatmentis preferably by nasal administration.

Tables 36-38 illustrate detection and measurement of TNFα, TNFR 1 andTNFR 2 in nasal mucus of patients with graded loss of smell followingtreatment with theophylline. Results indicate that detection andmeasurements of TNFα, TNFR 1 and TNFR 2 in nasal mucus can be used as anindex of the disease process and of changes toward normal as the diseaseis successfully treated, in the present case with theophylline. Further,nasal mucus can be used as an index of disease, disease severity andefficacy of disease treatment. Without limiting the scope of the presentinvention, this approach is applicable to other substances in nasalmucus in relationship to other disease processes (e.g., cancer, stroke)and to follow-up of their treatment with any drug.

Treatment with Transcranial Magnetic Stimulation

Loss of taste and smell acuity (hypogeusia and hyposmia, respectively)with subsequent gustatory and olfactory distortions in the absence oforal or external olfactory stimuli [(phantageusia and phantosmia,respectively) labeled sensory distortions], are symptoms which may occurin some patients without other neurological or psychological disorders.

Transcranial magnetic stimulation (TCMS) use has been limited by lack ofobjective methods to measure efficacy of its application. One aspect ofthe invention includes method of treatment of patients with loss oftaste and/or smell (hypogeusia and/or hyposmia, respectively) withsubsequent gustatory and/or olfactory distortions (phantageusia and/orphantosmia, respectively) with repetitive TCMS (rTCMS) which improvedtheir sensory acuity and decreased their sensory distortions.

Increased CA VI secretion has been considered a marker for bothincreased taste and smell function. Thus, before and after rTCMS, CA VIactivity and other salivary proteins were measured in patients with bothsensory loss and presence of sensory distortions. Since CA VI is a zinccontaining glycose talloprotein, the salivary zinc and copperconcentrations were also measured to determine if changes in theseparameters correlated with changes in CA VI activity. The possibility ofthe changes in other salivary proteins was also investigated. Changes inerythrocyte CA I, II as well as concentrations of zinc and copper inboth erythrocytes and in blood plasma, were also measured.

Example 40 shows the study of ninety-three patients with hyposmia,hypogeusia, phantosmia and/or phantageusia before and after rTCMS.Measurements were made of activities of CA I, II in erythrocytes and ofCA VI, of concentrations of zinc and copper in parotid saliva, bloodserum, and erythrocytes and of appearance of proteins in saliva bySELDI-TOF mass spectrometry. Results showed that after rTCMS,significant increases occurred in CA I, II, CA VI, and in concentrationsof zinc and copper in blood plasma, erythrocytes and saliva. Salivaryproteins at m/z value of 21.5K with a repeating pattern at intervals of5K m/z were induced.

These results demonstrate the biochemical changes in specific enzymaticactivities and trace metal concentrations following rTCMS. These changesmay relate not only to several aspects of clinical abnormalities ofsensory function but also to other neurological disorders includingepilepsy, parkinsonism, Alzheimer disease, head injury and motor neurondisease. Example 41 shows efficacy of treatment with rTCMS for patientswith these cognitive impairments such as hypogeusia, hyposmia,phantageusia, and phantosmia.

Other Example of Drugs

Drugs that may be used in the methods of treatment of the presentinvention may be selected from the following, viz. vaccination, alcoholabuse preparations, drugs used for Alzheimer's disease, anesthetics,acromegaly agents, analgesics, antiasthmatics, anticancer agents,anticoagulants and antithrombotic agents, anticonvulsants, antidiabeticsantiemetics, antiglaucoma, antihistamines, anti-infective agents,antiparkinsons, antiplatelet agents, antirheumatic agents,antispasmodics and anticholinergic agents, antitussives, carbonicanhydrase inhibitors, cardiovascular agents, cholinesterase inhibitors,treatment of CNS disorders, CNS; stimulants, contraceptives, cysticfibrosis management, dopamine receptor agonists, endometriosismanagement, erectile dysfunction therapy, fertility agents,gastrointestinal agents, immunomodulators and immunosuppressives, memoryenhancers, migraine preparations, muscle relaxants, nucleosideanalogues, osteoporosis management, parasympathomimetics,prostaglandins, psychotherapeutic agents, sedatives, hypnotics andtranquilizers, drugs used for slain ailments, steroids and hormones;Examples of alcohol abuse preparations are chlorazepate,chlordiazepoxide, diazepam, I disulfuram, hydroxyzine, naltrexone andtheir salts.

Examples of analgesics are acetaminophen, aspirin, bupivacain,boprenorphine, butorphanol, celecoxib, clofenadol, choline, clonidine,codeine, diflunisal, dihydrocodeine, dihydroergotamine, dihydromorphine,ethylmorphine, etodolac, eletriptan, eptazocine, ergotamine, fentanyl,fentoprofen, hyaluronic acid, hydrocodon, hydromorphon, hylan,ibuprofen, lindomethacin, ketorolac, lcetotifen, levomethadon,levallorphan, levorphanol, lidocaine, mefenamic acid, meloxicam,meperidine, metll adone, morphine, nabumetone, nalbuphin, nefopam,nalorphine, naloxone, naltrexone, naproxen, naratriptan, nefazodone,mormethadon, oxaprozin, oxycodone, oxymorphon, pentazocin, pethidine,phenpyramid, piritramid, piroxicam, propoxyphene, refecoxib,rizatriptan, salsalaketoprofen, sulindac, sumatriptan, tebacon, tilidin,tolmetin, tramadol, zolmitriptan and their salts.

Examples of antiasthmatics are ablukast, azelastine, bunaprolast,cinalukast, cromitrile, cromolyn, enofelast, isamoxole, ketotifen,levcromekalin, lodoxamide, montelukast, ontazolast, oxarbazole,oxatomide, piriprost potassium, pirolate, pobilukast edamine, quazolast,repirinast, ritolukast, sulukast, tetrazolastmeglumine, tiaramide,tibenelast, tomelukast, tranilast, verlukast, verofylline, szarirlukast.

Examples of anticancer agents are adriamycin, aldesleukin, allopurinol,altretamine, amifostine, anastrozole, asparaginase, betamethasone,bexarotene, bicalutamide, bleomycin, busulfan, capecitabine,carboplatin, cannustine, chlorambucil, cisplatin, cladarabine,conjugated estrogen, cortisone, cyclophosphamide, cylarabine,dacarbazine, daunorubicin, dactinomycin, denileukin, dexamethasone,discodermolide, docetaxel, doxorubicin, eloposidem, epirubicin, epoetin,epothilones, estramustine, esterified estrogen, ethinyl estradiol,etoposide, exemestane, flavopirdol, fluconazole, fludarabine,fluorouracil, flutamide, floxuridine, gemcitabine, gemtuzumab,goserelin, hexamethylmelamine, hydrocortisone, hydroxyurea, idarubicin,ifosfamide, interferon, irinotecan, lemiposide, letrozole, leuprolide,levamisole, levothyroxine, lomustine, mechlorethamine, melphalan,mercaptopurine mechlorethamine, megesterol, methotrexate,methylprednisolone, methyltestosterone, mithramycin, mitomycin,mitotane, mitoxantrone, mitozolomide, mutamycin, nilutamide, paclitaxel,pamidronate, pegaspargase, pentostatin, plicamycin, porfimer,prednisolone, procarbazine, rituximab, sargramostim, semustine,skeptozocin, tamoxifien, temozolomide, teniposide, testolactone,thioguanine, thiotepa, tomudex, topotecan, toremifene, trastumuzab,tretinoin, semustine, skeptozolocin, valrubicin, verteporfin,vinblastine, vincristine, vindesine, vinorelbine and their salts.

Examples of anticoagulants and antithrombic agents are warfarin,dalteparin, heparin, tinzaparin, enoxaparin, danaparoid, abciximab,alprostadil, altiplase, anagralide, aniskeplase, argatroban, ataprost,beraprost, camonagreel, cilostazol, clinprost, clopidogrel, cloricromen,dermatan, desirudin, domitroban, drotaverine, epoprostenol,eptifibatide, gabexate, iloprost, isbogrel, lamifiban, lamoteplase,lepirudin, levosimendan, lexipafant, melagatran, nafagrel, nafamostsat,nizofenone, orbifiban, ozagrel, pamicogrel, parnaparin, quinobendan,reteplase, sarpogralate, satigrel, silteplase, simendan, ticlopidine,vapiprost, tirofiban, xemilofiban, Y20811 and their salts.

Examples of anticonvulsants are carbamazopine, clonazepam, clorazepine,diazepam, divalproex, ethosuximide, ethotion, felbamate, fosphenyloin,gabapentin, lamotrigine, levetiracetam, lorazepam, mephenyloin,mephobarbital, metharbital, methsuximide, oxcarbazopine, phenobarbital,phenyloin, primidone, tiagabine, topiramate, valproic acid, vigabatrin,zonisamide, and their salts. Examples of antidiabetic agents areacarbose, acetohexamide, carbutamide, chlorpropamide, epalrestat,glibornuride, gliclazide, glimepiride, glipizide, gliquidone,glisoxepid, glyburide, glyhexamide, metformin, miglitol, nateglinide,orlistat, phenbutamide, pioglitazone, repaglinide, rosiglitazone,tolazamide, tolbutamide, tolcyclamide, tolrestat, troglitazone,voglibose and their salts.

Examples of antiemetics are alprazolam benzquinamide, benztropine,betahistine, chlorpromazine, dexamethasone, difenidol, dimenhydrinate,diphenhydramine, dolasetron, domperidone, dronabinol, droperidol,granisetron, haloperidol, lorazepam, meclizine, methylprednisolone,metoclopramide, ondansetron, perphenazine, prochlorperazine,promethazine, scopolamine, tributine, triethylperazine, triflupromazine,trimethobenzamide, tropisetron and their salts.

Examples of antiglaucoma agents are alprenoxime, dapiprazole,dipivefrin, latanoprost, naboctate, pirnabine and their salts.

Examples of antihistamines are acrivastine, activastine, albuterol,azelastine, bitolterol, alimemazine, amlexanox, azelastine, benzydamine,brompheniramine, cetirizine, chlorpheniramine, cimetidine, clemastine,cycloheptazine, cyproheptadine, diclofenac, diphenhydramine, dotarizine,ephedrine, epinastine, epinephrine, ethyluorepinephrine, fenpoterol,fexofenadine, flurbiprofen, hydroxyzine, ibuprofen, isoetharine,isoproterenol, ipratropium bromide, ketorolac, levocetirizine,loratidine, mequitazine, metaproterenol, phenylephrine, phenylpropanolamine, pirbuterol, promethazine, pseudo ephedrine, pyrilamine,salmeterol, terbutaline, tranilast, xanthine derivatives, xylometazolineand their salts.

Examples of anti-infective agents are abacavir, albendazole, amantadine,amphotericin, amikacin, aminosalicylic acid, amoxycillin, ampicillin,amprenavir, atovaquin, azithromycin, aztreonam, carbenicillin, cefaclor,cefadroxil, cefamandole, cefazolin, cefdinir, cefepime, cefexime,cefoperazone, cefotaxime, cefotitam, cefoperazone, cefoxitin,ceLpodoxine, cefprozil, ceftazidime, ceftibuten, ceftizoxime,ceftriaxone, cefuroxime, cephalexin, chloroquine, cidofovir, cilastatin,ciprofloxacin, clarithromycin, clavulinic acid, clindamycin,colistimethate, dalfopristine, dapsone, daunorubicin, delavirdin,demeclocycline, didanosine, doxycycline, doxorubicin, efavirenz,enoxacin, erythromycin, ethambutol, ethionamide, famsiclovir,fluconazole, flucytocin, foscarnet, fosfomycin, ganciclovir,gatifloxacin, griseofulvin, hydroxychloroquine, imipenem, indinavir,interferon, isoniazide, itraconazole, ivermectin, ketoconazole,lamivudine, levofloxacin, linezolide, lomefloxacin, lovacarbef,mebendazole, mefloquine, meropenem, methanamine, metronidazole,minocycline, moxefloxacin, nalidixic acid, nelfnavir, neomycin,nevirapine, nitrofurantoin, norfloxacin, ofloxacin, olseltamnivir,oxytetracycline, palivizumab, penicillins, perfloxacin, piperacillin,praziquantel, pyrazinamide, pyrimethamine, quinidine, quinupristine,retonavir, ribavirin, rifabutine, rifampicin, rimantadine, saquinavir,sparfloxacin, stavudine, streptomycin, sulfamethoxazole, teramycin,terbinafine, tetracycline, ticarcillin, thiabendazole, tobramycin,trimethoprim, trimetraxate, troleandomycin, trovafloxacin, valacyclovir,vancomycin, zalcitabine, zanamivir, zidovudine and their salts.

Examples of antiparkinsons are amantadine, adrogolide, altinicline,benztropine, biperiden, brasofensine, bromocriptine, budipine,cabergoline, dihydrexidine, entacapone, etilevodopa, idazoxan,iometopane, lazabemide, melevodopa, carbidopa/levodopa, mofegiline,moxiraprine, pergolide, pramipexole, quinelorane, rasagiline,ropinirole, seligiline, talipexole, tolcapone, trihexyphenidyl and theirsalts. Examples of antirheumatic agents are azathiprine, betamethasone,celecoxib, cyclosporin, diclofenac, hydroxychloroquine, indomethacin,infliximab, mercaptobutanedioic acid, methylprednisolone, naproxen,penicillamine, piroxicam, prednisolone, sulfasalazine and their salts.

Examples of platelet agents are abciximab, anagrelide, aspirin,cilostazol, clopidogrel, dipyridamole, epoprostenol, eptifbatide,ticlopidine, tinofban and their salts. Examples of antispasmodics andanticholinergic agents are aspirin, atropine, diclofenac, hyoscyamine,mesoprostol, methocarbamol, phenobarbital, scopolamine and their salts.

Examples of antitussives are acetaminophen, acrivastin, albuterol,benzonatate, beractant, brompheniramine, caffeine, calfactant,carbetapentane, chlorpheniramine, codeine, colfuscerin,dextromethorphan, dornase alpha, doxylamine, epinephrine, fexofenadine,guaiphenesin, iprakopium, levalbuterol, metaproterenol, montelukast,pentoxyphyline, phenylephrine, phenylpropanolamine, pirbuterol,poractant alpha, pseudoephedrine, pyrilamine, salbuterol, salmeterol,terbutaline, theophylline, zafirlukast, zileuton and their salts.Examples of carbonic anhydrase inhibitors are acetazolamide,dichlorphenamide, dorzolamide, methazolamide, sezolamide and theirsalts.

Examples of cardiovascular agents are abciximab, acebutolol, activase,adenosine, adrenaline, amidarone, amiloride, amlodipine, amyl nikate,atenolol, atorvastatin, benazepril, bepiridil, betaxalol, bisoprolol,candesartan, captopril, cartenolol, carvedilol, cerivastatin,chlorthalidone, chlorthiazole, clofibrate, clonidine, colestipol,colosevelam, digoxin, diltiazem, disopyramide, dobutamine, dofetilide,doxazosin, enalapril, epoprostenol, eprosartan, esmolol, ethacrynate,erythrityl, felodipine, fenoidapam, fosinopril, fleicamide,fluorosemide, fluvastatin, gewhibrozil, hydrochlorthiazide,hydroflumethazine, ibutilide, indapamide, isosorbide, irbesartan,labetolol, lacidipine, lisinopril, losartan, lovastatin, mecamylamine,metoprolol, metaraminol, metazolone, methylchlorthiazide, methyldopa,metyrosine, mexiletine, midrodine, milrinone, moexipril, nadolol,niacin, nicardipine, nicorandil, nifedipine, nimodipine, nisoldipine,nikoglycerin, phenoxybenzamine, perindopril, polythiazide, pravastatin,prazosin, procainamide, propafenone, propranolol, quanfacine, quinapril,quinidine, ranipril, reteplase, simvastatin, sotalol, spironolactone,skeptokinase, telmisartan, terazosin, timolol, tocainamide, tors-emide,kandolapril, kiamterene, kapidil, valsartan and their salts.

Examples of cholinesterase inhibitors are donepezil, edrophonium,neostigmine, pyridostigmine, rivasti.gmine, tacrine and their salts.Examples of CNS stimulants are caffeine, doxapram, dexoamphetamine,donepezil, edrophonium, methamphetamine, methylphenidate, modafinil,neostigwine, pemoline, phentermine, pyridostigmine, rivastigwine, tacrinand their salts. Examples of cystic fibrosis management are dornasealpha, pancrelipase, tobramycin and their salts. Examples of dopaminereceptor agonists are amantadine, cabergoline, fenoldopam, pergolide,pramipexil, ropinirole and their salts. Examples of drugs used forendometriosis management are danazol, goserelin, leuprolide, nafarelin,norethindrone and their salts. Examples of drugs used for erectiledysfunction therapy are alprostadil, sildenafil, yohimbine and theirsalts. I Examples of gastrointestinal agents are aldosetron, bisacodyl,bismuth subsalicylate, celecoxib, difoxin, dipheoxylate, docusate,famotidine, glycopyrrolate, infliximab, lansoprazole, loperamide,metaclopramide, nizatidine, omeprazole, pantoprazole, rabeprazole,ranitidine, simethicone, sucralfate, and their salts.

Examples of immunomodulators and immunosupressives are azathioprin,ceftizoxine, cyclosporin, daclizumab, glatiramer, immunoglobulin,interferon, leflunomide, levamisol, mycophenolate, mausomanab,phthalidomide, ribavirin, sirolimus and their salts. Examples of drugsused in Alzheimer's disease are donepezil, galanthamine, metrifonate,rivastigwine, tacrine, TAK-147 and their salts. Examples of drugs usedfor migraine preparations are acetaminophen, dihydroergotamine,divalproex, ergotamine, propranolol, risatriptan, sumatriptan,trimetrexate and their salts. Examples of muscle relaxants arealcuronium-chloride, azapropazon, atracurium, baclofen, carisoprodol,quinine derivatives, chloromezanon, chlorophenesincarbamate,chlorozoxazon, cyclobenzaprine, dantrolene, decamethoniumbromide,dimethyltubocurariniumchloride, doxacurium, fenyrami dol, gallamintriethio dide, guaiphenesin, hexafluoreniumbromide,hexacarbacholinbromide, memantin, mephenesin, meprobamate, metamisol,metaxalone, methocarbamol, mivacurium, orphenadrin, pancuronium,phenazon, phenprobamate, pip e curonium, rap acuronium, ro curonium, succinylcholine, soxamethoniumchloride, tetrazepam, tizanidine,tubocurarine chloride, tybamate, vecuronium and their salts.

Examples of nucleoside analogues are abacavir, acyclovir, didanosine,ganciclovir, gewcitabine, lamivudine, ribavirin, stavudine, zalcitabineand their salts. Examples of drugs used for osteoporosis management arealendronate, calcitonin, estradiol, estropipate, medroxyprogesterone,norethindrone, norgestimate, pamidronate, raloxifen, risdronate,zolendronate and their salts. Examples of parasympathomimetics arebethanechol, piperidine, edrophonium, glycopyrolate, hyoscyamine,pilocarpine, tacrine, yohimbine and their salts. Examples ofprostaglandins are alprostadil, epoprostenol, misoprostol and theirsalts. Examples of psychotherapeutic agents are acetophenazine,alentemol, alpertine, alprazolam, amitriptyline, aripiprazole,azaperone, batelapine, befipiride, benperidol, benzindopyrine, bimithil,biriperone, brofoxine; bromperidol; bupropion, buspirone, butaclamol,butaperazine; carphenazine, carvotroline, cericlamine, chlorazepine,chlordiazepoxide, chlorpromazine; chlorprothixene, cinperene,cintriamide, citalopram, clomacran, clonazopam, clopenthixol,clopimozide, clopipazan, cloroperone, clothiapine, clothixamide,clozapine; cyclophenazine, dapiprazole, dapoxetine, desipramine,divalproex, dipyridamole, doxepin, droperidol, duloxetine, eltoprazine,eptipirone, etazolate, fenimide, fibanserin, flucindole, flumezapine,fluoxetine, fluphenazine, fluspiperone, fluspirilene, flutroline,fluvoxamine, gepione, gevotroline, halopemide, haloperidol, hydroxyzine,hydroxynortriptyline, iloperidone, imidoline, lamotrigine, loxapine,enperone, mazapertine, mephobarbital, meprobamate, mesoridazine,mesoridazine, milnacipran, mirtazapine, metiapine, milenperone,milipertine, molindone, nafadotride, naranol, nefazodone, neflumozide,ocaperidone, odapipam, olanzapine, oxethiazine, oxiperomide, pagoclone,paliperidone, paroxitene, penfluridol, pentiapine perphenazine,phenelzine, pimozide, pinoxepin, pipamperone, piperacetazine,pipotiazine, piquindone, pilindole, pivagabine, pramipexole,prochlorperazine, prochlorperazine, promazine, quetiapine, reboxetine,remoxipride, remoxipride, risperidone, rimcazole, robolzotan,selegiline, seperidol, sertraline, sertindole; seteptiline, setoperone,spiperone, sunipitron, tepiindole, thioridazine, thiothixene, tiapride,tioperidone, tiospione, topiramate, tranylcypromine, trifluoperazine,trifluperidol, triflupromazine, triflupromazine, kimipramine,venlafaxine, ziprasidone and their salts.

Examples of sedatives, hypnotics and tranquilisers are bromazepam,buspione, clazolam, clobazam, chlorazepate, diazepam, demoxepam,dexmedetomitine, diphenyhydramine, doxylamine, enciprazine, estrazolam,hydroxyzine, ketazolam, lorazatone, lorazepam, loxapine, medazepam,meperidine, methobarbital, midazolam, nabilone, nisobamate, oxazepam,pentobarbital, promethazine, propofol, triazolam, zalelplon, zolpidemand their salts. Examples of drugs used for treatment of skin ailmentsare acitretin, alclometasone, allitretinoin, betamethasone,calciprotrine, chlorhexidine, clobetasol, clocortolone, clotriamozole,collagenase, cyclosporin, desonide, difluorosone, doxepine,eflornithine, finasteride, fluocinolone, flurandrenolide, fluticasone,halobetasol, hydrochloroquine, hydroquinone, hydroxyzine, ketoconazole,mafenide, malathion, menobenzone, neostigmine, nystatin, podoflox,povidone, tazorotene, tretinoin and their salts.

Examples of steroids and hormones are alclometasone, betamethasone,calcitonin, cikorelix, clobetasol, clocortolone, cortisones, danazol,desmopressin, desonide, desogestrel, desoximetasone, dexamethasone,diflorasone, estradiol, estrogens, estropipate, ethynlestradiol, ifluocinolone, flurandrenolide, fluticasone, glucagon, gonadotropin,goserelin, halobetasol, hydrocortisone, leuprolide, levonorgestrel,levothyroxine, medroxyprogesterone, menotropins, methylprednisolone,methyltestosterone, mometasone, naferelin, norditropin, norethindrone,norgestrel, octreolide, oxandrolone, oxymetholone, polytropin,prednicarbate, prednisolone, progesterone, sermorelin, somatropin,stanozolol, testosterone, urofollitropin and their salts.

Example of agents that are susceptible to the gastric environment suchas proton pump inhibitors are pantoprazole, omeprazole, lansoprazole,esomeprazole, rabeprazole, pariprazole, leminoprazole, or an enantiomer,isomer, derivative, free base or salt thereof; lipid-lowering agentssuch as lovastatin, pravastatin, atorvastatin, simvastatin; agents thatare targeted to the intestine for local action such as 5-aminosalicylicacid, corticosteroids such as beclomethasone, budesonide, fluticasone,tixocortol useful in treating Crohn's disease and ulcerative colitis;agents that may be inactivated by the gastric contents such as enzymeslike pancreatin, antibiotics such as erythromycin; agents that causebleeding or irritation of the gastric mucosa such as aspirin, steroids,non-steroidal anti-inflammatory compounds like ibuprofen, naproxen,ketoprofen, fenoprofen, flurbiprofen, oxaprozin, diflunisal, diclofenac,indomethacin, tolmetin, sulindac, etodolac, acetaminophen, plateletinhibitors such as abciximab, intergrelin, dipyridamole; nucleosideanalogs such as didanosine, transfer factor preparations, hormones,insulin, and other agents that have decreased stability in the gastricenvironment, as well as agents that are required for local action in thelatter part of the gastrointestinal tract. The agents may be used astheir base or as their pharmaceutically acceptable salt or solvatethereof.

The treatment can be via oral administration, transmucosaladministration, buccal administration, nasal administration, inhalation,parental administration, intravenous, subcutaneous, intramuscular,sublingual, transdermal administration, and rectal administration. Nasaladministration is a preferred mode in the present invention. Nasaladministration may delay or obviate drug resistance that may occurthrough the other routes of administration, such as, oral or parentral.

Effective Dosages

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredient is contained in atherapeutically or prophylactically effective amount, i.e., in an amounteffective to achieve therapeutic or prophylactic benefit. Of course, theactual amount effective for a particular application will depend, interalia, on the condition being treated and the route of administration.Determination of an effective amount is well within the capabilities ofthose skilled in the art, especially in light of the disclosure herein.

Therapeutically effective amounts for use in humans can be determinedfrom animal models. For example, a dose for humans can be formulated toachieve circulating concentration that has been found to be effective inanimals. The amount administered can be the same amount administered totreat a particular disease or can be an amount lower than the amountadministered to treat that particular disease. Patient doses for oraladministration of the drug may range from about 1 μg-1 gm/day. Thedosage may be administered once per day or several or multiple times perday. The amount of the drug administered to practice methods of thepresent invention will of course, be dependent on the subject beingtreated, the severity of the affliction, the manner of administrationand the judgment of the prescribing physician. The dose used to practicethe invention can produce the desired therapeutic or prophylacticeffects, without producing serious side effects.

Routes of Administration

The methods of treatment in the invention include by way of exampleonly, oral administration, transmucosal administration, buccaladministration, nasal administration such as inhalation, parentaladministration, intravenous, subcutaneous, intramuscular, sublingual,transdermal administration, and rectal administration.

In some embodiments of the present invention, the method of treatment isby nasal administration or inhalation. Compositions for inhalation orinsufflation include solutions and suspensions in pharmaceuticallyacceptable, aqueous or organic solvents, or mixtures thereof, andpowders. The liquid or solid compositions may contain suitablepharmaceutically acceptable excipients as described supra. Thecompositions can be administered by the oral or nasal respiratory routefor local or systemic effect. Compositions in preferablypharmaceutically acceptable solvents may be nebulized by use of inertgases. Nebulized solutions may be inhaled directly from the nebulizingdevice or the nebulizing device may be attached to a face mask tent, orintermittent positive pressure breathing machine. Solution, suspension,or powder compositions may be administered, preferably orally ornasally, from devices that deliver the formulation in an appropriatemanner.

In some embodiments of the present invention, the method of treatment isby oral administration. Oral administration can be presented as discretedosage forms, such as capsules, cachets, or tablets, or liquids oraerosol sprays each containing a predetermined amount of an activeingredient as a powder or in granules, a solution, or a suspension in anaqueous or non-aqueous liquid, an oil-in-water emulsion, or awater-in-oil liquid emulsion. Such dosage forms can be prepared by anyof the methods of pharmacy, but all methods include the step of bringingthe active ingredient into association with the carrier, whichconstitutes one or more necessary ingredients. In general, thecompositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product into the desiredpresentation. For example, a tablet can be prepared by compression ormolding, optionally with one or more accessory ingredients. Compressedtablets can be prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as powder or granules, optionallymixed with an excipient such as, but not limited to, a binder, alubricant, an inert diluent, and/or a surface active or dispersingagent. Molded tablets can be made by molding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.

An active ingredient can be combined in an intimate admixture with apharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier can take a wide variety of formsdepending on the form of preparation desired for administration. Inpreparing the compositions for an oral dosage form, any of the usualpharmaceutical media can be employed as carriers, such as, for example,water, glycols, oils, alcohols, flavoring agents, preservatives,coloring agents, and the like in the case of oral liquid preparations(such as suspensions, solutions, and elixirs) or aerosols; or carrierssuch as starches, sugars, micro-crystalline cellulose, diluents,granulating agents, lubricants, binders, and disintegrating agents canbe used in the case of oral solid preparations, in some embodimentswithout employing the use of lactose. For example, suitable carriersinclude powders, capsules, and tablets, with the solid oralpreparations. If desired, tablets can be coated by standard aqueous ornonaqueous techniques.

Examples of suitable fillers for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.

Disintegrants may be used in the method of treatment of the presentinvention to provide tablets that disintegrate when exposed to anaqueous environment. Disintegrants that can be used to formpharmaceutical compositions and dosage forms of the invention include,but are not limited to, agar-agar, alginic acid, calcium carbonate,microcrystalline cellulose, croscarmellose sodium, crospovidone,polacrilin potassium, sodium starch glycolate, potato or tapioca starch,other starches, pre-gelatinized starch, other starches, clays, otheralgins, other celluloses, gums or mixtures thereof.

Lubricants which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, ormixtures thereof. Additional lubricants include, for example, a syloidsilica gel, a coagulated aerosol of synthetic silica, or mixturesthereof. A lubricant can optionally be added, in an amount of less thanabout 1 weight percent of the pharmaceutical composition.

When aqueous suspensions and/or elixirs are desired for oraladministration, the essential active ingredient therein may be combinedwith various sweetening or flavoring agents, coloring matter or dyesand, if so desired, emulsifying and/or suspending agents, together withsuch diluents as water, ethanol, propylene glycol, glycerin and variouscombinations thereof.

The tablets can be uncoated or coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate canbe employed. Formulations for oral use can also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin, or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, for example, peanut oil, liquidparaffin or olive oil.

Surfactant which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to,hydrophilic surfactants, lipophilic surfactants, and mixtures thereof.That is, a mixture of hydrophilic surfactants may be employed, a mixtureof lipophilic surfactants may be employed, or a mixture of at least onehydrophilic surfactant and at least one lipophilic surfactant may beemployed. A solubilizer may also be added to increase the solubility ofthe hydrophilic drug and/or other components, such as surfactants, or tomaintain the composition as a stable or homogeneous solution ordispersion.

Mixtures of solubilizers may be used. Examples include, but not limitedto, triacetin, triethylcitrate, ethyl oleate, ethyl caprylate,dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone,polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropylcyclodextrins, ethanol, polyethylene glycol 200-100, glycofurol,transcutol, propylene glycol, and dimethyl isosorbide. Particularlypreferred solubilizers include sorbitol, glycerol, triacetin, ethylalcohol, PEG-400, glycofurol and propylene glycol.

The compositions for the treatment can further include one or morepharmaceutically acceptable additives and excipients. Such additives andexcipients include, without limitation, detackifiers, anti-foamingagents, buffering agents, polymers, antioxidants, preservatives,chelating agents, viscomodulators, tonicifiers, flavorants, colorants,odorants, opacifiers, suspending agents, binders, fillers, plasticizers,lubricants, and mixtures thereof.

In addition, an acid or a base may be incorporated into the compositionto facilitate processing, to enhance stability, or for other reasons.Examples of pharmaceutically acceptable bases include amino acids, aminoacid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide,sodium hydrogen carbonate, aluminum hydroxide, calcium carbonate,magnesium hydroxide, magnesium aluminum silicate, synthetic aluminumsilicate, synthetic hydrocalcite, magnesium aluminum hydroxide,diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine,triethylamine, triisopropanolamine, trimethylamine,tris(hydroxymethyl)aminomethane (TRIS) and the like. Suitable acids arepharmaceutically acceptable organic or inorganic acids. Examples ofsuitable inorganic acids include hydrochloric acid, hydrobromic acid,hydriodic acid, sulfuric acid, nitric acid, boric acid, phosphoric acid,and the like. Examples of suitable organic acids include acetic acid,acrylic acid, adipic acid, alginic acid, alkanesulfonic acids, aminoacids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonicacid, citric acid, fatty acids, formic acid, fumaric acid, gluconicacid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleicacid, methanesulfonic acid, oxalic acid, para-bromophenylsulfonic acid,propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid,succinic acid, tannic acid, tartaric acid, thioglycolic acid,toluenesulfonic acid, uric acid and the like.

The forms in which the compositions of the present invention may beincorporated for administration by injection include aqueous or oilsuspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, orpeanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueoussolution, and similar pharmaceutical vehicles.

Aqueous solutions in saline are also conventionally used for injection.Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and thelike (and suitable mixtures thereof), cyclodextrin derivatives, andvegetable oils may also be employed. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like.

The compositions for delivery can be formulated into preparations insolid, semi-solid, or liquid forms suitable for local or topicaladministration, such as gels, water soluble jellies, creams, lotions,suspensions, foams, powders, slurries, ointments, solutions, oils,pastes, suppositories, sprays, emulsions, saline solutions,dimethylsulfoxide (DMSO)-based solutions. In general, carriers withhigher densities are capable of providing an area with a prolongedexposure to the active ingredients. In contrast, a solution formulationmay provide more immediate exposure of the active ingredient to thechosen area.

In some embodiments of the present invention, the method of treatmentcan be transdermal. Transdermal patches may be used to providecontinuous or discontinuous infusion in controlled amounts, either withor without therapeutic agent. The construction and use of transdermalpatches for the delivery of pharmaceutical agents is well known in theart. See, e.g., U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Suchpatches may be constructed for continuous, pulsatile, or on demanddelivery of pharmaceutical agents.

Pharmaceutical compositions may also be prepared with one or morepharmaceutically acceptable excipients suitable for sublingual, buccal,rectal, intraosseous, intraocular, intranasal, epidural, or intraspinaladministration. Preparations for such pharmaceutical compositions arewell-known in the art. See, e.g., See, e.g., Anderson, Philip O.;Knoben, James E.; Troutman, William G, eds., Handbook of Clinical DrugData, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds.,Principles of Drug Action, Third Edition, Churchill Livingston, N.Y.,1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition,McGraw Hill, 20037ybg; Goodman and Gilman, eds., The PharmacologicalBasis of Therapeutics, Tenth Edition, McGraw Hill, 2001; RemingtonsPharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins., 2000;Martindale, The Extra Pharmacopoeia, Thirty-Second Edition (ThePharmaceutical Press, London, 1999); all of which are incorporated byreference herein in their entirety.

Kits

The invention also provides kits. As shown in FIG. 3, by way of exampleonly, the kit 300 may include a sterile nasal swab 301 for collection ofnasal secretions, an elongated storage and transport tube 302 forreceiving the swab wherein the tube can be glass or plastic and the tubemay have a replaceable end closure, and contain a sterile nutrientmedium for isolation of the nasal secretions, a sterile assay solution303 for addition to the transport tube, and a detector medium 304 forthe detection of a biological substance in the nasal secretion. The kitmay also include written instructions 305. In some embodiments, thetherapeutic agent can also be provided as separate compositions inseparate containers within the kit for the treatment. Suitable packagingand additional articles for use (e.g., measuring cup for liquidpreparations, foil wrapping to minimize exposure to air, and the like)are known in the art and may be included in the kit.

The following preparations and examples serve to illustrate theinvention. They should not be construed as narrowing it, or limiting itsscope.

EXAMPLES Example 1 PCR Analysis of a Specimen of Nasal Mucus

Specimen of nasal mucus from different subjects was collected andanalyzed using PCR. FIG. 4 depicts a polyacrylamide gel electrophoresisof samples as shown in Table 1. Lanes 1-7 in FIG. 4 reveal one majorband consistent with the presence of HLA. Lanes 8-14 reveal one majorband consistent with the presence of β globin. On the right are locatedmolecular weight markers of various KD.

TABLE 1 Results of PCR analysis of two samples of nasal mucus obtainedfrom normal subjects Area 3 (Units)* Rot. Rep. Sample Known Tm1 Area 1Tm2 Area 2 Tm3 Ethidium Sample Pos. Sample Name of . . . Type* Conc. (°C.) (Units) (° C.) (Units) (° C.) Bromide Comments 1 WB Pos Cont U 84.0511.18 Pos HLA 2 R. Blk U 82.08 10.96 Neg HLA 3 Specimen # 1 U 83.919.602 Pos HLA 4 Specimen # 2 U 84.74 10.86 Pos HLA 5 Specimen # 1 + U83.40 7.006 Pos HLA Pos Cont 6 Specimen # 1 + U 84.63 8.658 Pos HLA PosCont 7 R. Blk U 80.68 5.131 Neg HLA 8 WB Pos Cont U 87.41 6.062 Pos betaglobin 9 R. Blk U 75.92 6.166 Pos beta globin 10 Specimen # 1 U 86.977.534 Pos beta globin 11 Specimen # 2 U 76.91 5.492 86.91 8.009 Pos betaglobin 12 Specimen # 1 + U 86.81 7.983 Pos beta globin Pos Cont 13Specimen # 1 + U 86.54 7.451 Pos beta globin Pos Cont 14 R. Blk U Negbeta globin *P = Positive, U = Unknown, N = Negative, S = Standard, < >= De-Selected

Example 2 DNA Extraction Procedure from Body Fluids with QIAGEN Kit

All specimens and all reagents were equilibrated to room temperature. 20μl Qiagen Protease (or proteinase K) was pipetted into the bottom of a1.5 ml centrifuge tube. 200 μl of specimen (nasal mucus, plasma, saliva,urine and other biological fluids) was added to the centrifuge tube. Incase of specimens containing less than 1 μg of DNA or RNA, 5-10 μg ofcarrier DNA or RNA (20 μl of poly dA or 8 μl of poly [C]) was added. 200μl of AL buffer was added to the tube. Mixture was mixed bypulse-vortexing for 15 sec. Mixture was incubated at 56-60° C. for 15min. Mixture was vortexed and centrifuged briefly. 200 μl of ethanol(96-100%) was added to the tube. Mixture was vortexed and centrifugedbriefly. The mixture was carefully applied to a QIAamp spin column (in a2 ml collection tube) without wetting the rim. Caps of the columns wereclosed and the mixture was centrifuged for 1 min. The spin column wasplaced in another clean collection tube and the tube containing thefiltrate was discarded. 500 μl of buffer AW1 was added to the spincolumn, the lid was closed and the column was spun for 1 min. The columnwas placed in another clean collection tube and the tube containing thefiltrate was discarded. 500 μl of buffer AW2 was added to the spincolumn, the lid was closed and the column was spun at for 3 minutes; thecollection tube containing the filtrate was discarded. Since traceamounts of buffer AW2 inhibit PCR, complete removal of the buffer isdesirable. The column is then placed in a clean 1.5 ml centrifuge tubeand 50 μl of H₂O was added to it. It was incubated at room temperaturefor 15 minutes and centrifuged for 3 minutes. The resulting DNA solutioncan be stored at 4° C. for several months.

Example 3 LightCycler Data Analysis Report

FIG. 5 depicts LightCycler melting peak report on results of PCRanalysis of two samples of nasal mucus. Fluorescence is plotted onordinate, temperature on abscissa. FIG. 6 depicts LightCycler dataanalysis report on results of PCR analysis of two samples of nasalmucus. Fluorescence is plotted on ordinate, cycle number on abscissa.FIG. 7 depicts LightCycler melting peak report on results of PCRanalysis of two samples of nasal mucus. Fluorescence is plotted onordinate, temperature on abscissa. FIG. 8 depicts LightCycler dataanalysis report on results of PCR analysis of two samples of nasalmucus. Fluorescence is platted on ordinate, cycle number on abscissa.FIG. 9 depicts LightCycler data analysis report on results of PCRanalysis of two samples of nasal mucus. Fluorescence is plotted onordinate, cycle number on abscissa.

TABLE 2 LightCycler Melting Analysis Report of Studies on each of twosamples of nasal mucus analyzed by PCR Program: Denature Type: NoneCycles 1 Segment Temperature Hold Time Slope 2° Target Step Size StepDelay Acquisition Number Target (° C.) (sec) (C. °/sec) Temp (° C.) (°C.) (Cycles) Mode 1 95 600 20 0 0 0 None Program: PCR Type:Quantification Cycles 45 Segment Temperature Hold Time Slope 2° TargetStep Size Step Delay Acquisition Number Target (° C.) (sec) (C. °/sec)Temp (° C.) (° C.) (Cycles) Mode 1 95 10 20 0 0 0 None 2 58 15 20 0 0 0None 3 72 12 20 0 0 0 Single Program: melt Type: None Cycles 1 SegmentTemperature Hold Time Slope 2° Target Step Size Step Delay AcquisitionNumber Target (° C.) (sec) (C. °/sec) Temp (° C.) (° C.) (Cycles) Mode 195 0 20 0 0 0 None 2 50 60 20 0 0 0 None 3 95 0 0.2 0 0 0 ContinuousProgram: cool Type: None Cycles 1 Segment Temperature Hold Time Slope 2°Target Step Size Step Delay Acquisition Number Target (° C.) (sec) (C.°/sec) Temp (° C.) (° C.) (Cycles) Mode 1 40 30 20 0 0 0 NoneFlouresence Setings Melting Analysis Settings LED Power CALIB DisplayMode 3.5 Compatible Channel Setting F1/1 Color N/A Program Name meltCompensation N/A Start Time 0:52:39.85 Stop Time 0:56:28.2 Car. MovementContinuous

Example 4 Analysis of Nasal Mucus Before and after Fasting

Table 3 depicts the results of the ELISA analysis of nasal mucuscollected from 49 subjects before (fasting) and after (non-fasting).FL/VOL is flow rate in ml/min, PROT is protein, LEP is leptin, LEP/PR isleptin/protein, AG is agouti related protein, AG/PR is agouti relatedprotein/protein, INS is insulin, INS/PR is insulin/protein, X is meanvalues of 49 subjects, and SD is standard deviation of results.

TABLE 3 Nasal mucus (nM) BEFORE AFTER BEFORE AFTER BEFORE AFTER BEFOREAFTER FL/VOL FL/VOL PROT PROT LEP LEP LEP/PR LEP/PR SUBJECT ml/minml/min mg/ml mg/ml pg/ml pg/ml ratio ratio 1 6.52 2.198 496 226 2 0.812.771 90 3 0.31 2.016 39 4 3.27 2.494 5 4.64 2.811 724 258 6 0.28 4.033.237 2.419 197 61 7 49.31 29.68 2.886 2.805 510 382 177 136 8 5.183.272 9 22.37 4.76 1.751 1.999 10 3 1.653 11 0.39 3.865 56 14 12 3.831.192 13 18.38 3.047 230 75 14 0.27 2365 15 1.37 2.834 16 8.36 2.252 3415 17 9.9 2.644 28 11 18 29.13 21.63 3.859 3.093 45 51 12 16 19 2.931.584 18 11 20 1.32 2.114 21 5.84 2.068 22 5.94 1.63 2.16 2.673 107 5023 3 3.22 24 2.72 2.246 39 17 25 3.87 4.87 2.28 2.339 39 17 26 1.992.707 174 64 27 27.24 2.845 85 30 28 3 2.362 79 33 29 2.21 1.14 30 1.113.226 31 5.59 2.39 32 6.77 2.811 129 46 33 0.55 3.122 388 124 34 1.022.062 56 27 35 23.8 1.665 56 34 36 1.13 2.92 225 77 37 1.27 1.323 39 2938 3.33 3.537 45 13 39 6.39 1.901 287 151 40 1.27 1.356 135 100 41 1.222.379 183 77 42 3.35 2.72 2.609 2.892 73 107 28 37 43 10.23 1.123 124110 44 13.07 10.58 3.531 1.74 540 1265 153 727 45 4.15 2.552 22 9 462.76 3.83 3.012 3.859 107 124 36 32 47 3.34 3.37 143 42 48 10.42 3.233.335 2.419 104 56 31 23 49 1.09 2.016 X 7.21 6.77 2.55 2.46 276.0 182.270.5 95.9 SD 10.23 7.65 0.71 0.68 475.0 296.1 69.7 180.3 BEFORE AFTERBEFORE AFTER AG AG BEFORE AFTER INS INS BEFORE AFTER SUBJECT pg/ml pg/mlAG/PR AG/PR μIU/ml μIU/ml INS/PR INS/PR 1 1 0 22 10 2 3 4 7 3 10 4 5 7 27 2 6 1 8 0 3 5 16 2 7 7 13 7 5 2 8 7 2 31 9 9 2 1 15 5 9 3 10 7 4 23 1411 6 2 8 2 12 1 1 6 5 13 4 1 10 3 14 15 5 2 1 0 16 17 18 9 11 2 4 4 12 14 19 2 1 19 12 20 8 4 11 5 21 3.5 2 15 7 22 3 2 1 1 11 16 5 6 23 7 2 9 324 7 3 8 4 25 21 8 9 3 43 48 19 21 26 5 2 9 3 27 7 2 9 3 28 7 3 43 18 299 8 8 7 30 8 2 31 10 31 7 3 74 31 32 33 1 0 95 30 34 35 3 2 13 8 36 8 319 7 37 38 39 6 3 40 6 4 27 20 41 0 0 25 11 42 2 9 1 3 4 12 2 4 43 3 3 22 44 2 1 51 24 14 14 45 16 6 46 6 10 2 3 14 15 5 4 47 7 2 10 3 48 4 3 11 4 6 1 2 49 6 3 X 5.5 6.1 2.3 2.6 18.6 18.0 7.5 8.1 SD 4.0 3.0 1.9 1.220.6 11.8 7.5 6.4

Example 5 Analysis of Saliva Before and after Fasting

Table 4 depicts the results of the ELISA analysis of saliva collectedfrom 50 subjects before (fasting) and after (non-fasting). FL/VOL isflow rate in ml/min, PROT is protein, LEP is leptin, LEP/PR isleptin/protein, AG is agouti related protein, AG/PR is agouti relatedprotein/protein, INS is insulin, INS/PR is insulin/protein, X is meanvalues of 50 subjects, and SD is standard deviation of results.

TABLE 4 Saliva BEFORE AFTER BEFORE AFTER BEFORE AFTER FL/VOL FL/VOL PROTPROT LEP LEP BEFORE AFTER SUBJECT ml/min ml/min mg/ml mg/ml pg/ml pg/mlLEP/PR LEP/PR 1 0.456 2.483 10 4.03 2 0.61 2.546 7 2.75 3 0.673 2.684 62.24 4 0.406 1.941 5 0.717 3.623 24 6.62 6 0.447 0.405 2.523 2.886 176.74 7 0.572 1.119 3.946 3.491 2 4 0.51 1.15 8 0.405 3.473 9 0.315 2.8172.177 10 0.377 2.39 11 0.84 3.197 12 3.75 12 0.635 3.594 10 2.78 130.721 3.306 8 2.42 14 0.908 3.433 5 1.46 15 0.908 3.277 16 0.523 3.508 51.43 17 0.5 2.85 6 2.11 18 0.633 0.722 3.214 3.254 6 15 1.87 4.61 190.356 3.3 19 5.76 20 0.734 1.855 7 3.77 21 0.692 3.398 22 0.584 3.646 231.211 0.705 3.145 3.295 11 3.50 24 0.601 2.483 25 0.634 1.377 12 8.71 260.347 0.372 2.736 2.932 16 5.85 27 0.356 2.413 6 2.49 28 0.559 3.012 237.64 29 0.871 3.076 20 6.50 30 0.722 2.091 31 0.946 3.819 32 0.355 2.98933 0.496 3.669 11 3.00 34 0.569 3.456 16 4.63 35 0.34 3.358 12 3.57 360.766 2.033 15 7.38 37 0.969 3.024 6 1.98 38 0.824 3.427 22 6.42 390.624 2.31 7 3.03 40 0.71 1.711 3 1.75 41 0.905 2.644 8 3.03 42 0.5112.483 25 10.07 43 0.524 0.614 2.776 3.56 7 10 2.52 2.81 44 0.533 3.185 61.88 45 0.596 0.566 3.963 3.128 6 7 1.51 2.24 46 0.806 2.068 7 3.38 471.041 0.908 3.295 2.748 11 17 3.34 6.19 48 0.714 2.863 0 0.00 49 0.5240.631 3.4 2.638 10 19 2.94 7.20 50 0.841 2.65 X 0.64 0.64 2.93 2.96 11.49.8 4.1 3.3 SD 0.21 0.21 0.62 0.53 6.7 5.5 2.6 1.8 BEFORE AFTER BEFOREAFTER AG AG BEFORE AFTER INS INS BEFORE AFTER SUBJECT pg/ml pg/ml AG/PRAG/PR μIU/ml μIU/ml INS/PR INS/PR 1 15 6.04 17 6.85 2 3 4 6 3.09 2010.30 5 8 2.21 21 5.80 6 6 8 2.38 2.77 15 12 5.95 4.16 7 9 9 2.28 2.58 82 0.58 10 2.88 9 8 3.67 6 4 2.13 1.84 10 7 2.93 28 11.72 11 15 4.69 113.44 12 7 1.95 15 4.17 13 2 0.60 19 5.75 14 79 15 13 3.97 27 16 17 18 45 1.24 1.54 20 11 6.22 3.38 19 31 9.39 7 2.12 20 25 13.48 21 10 2.94 102.94 22 46 12.62 43 11.79 23 6 8 1.91 2.43 13 2 4.13 0.61 24 8 3.22 208.05 25 7 5.08 27 19.61 26 10 7 3.65 2.39 9 2 3.29 0.68 27 5 2.07 9 3.7328 8 2.66 17 5.64 29 7 2.28 12 3.90 30 10 4.78 44 21.04 31 4 1.05 277.07 32 5 1.67 23 7.69 33 34 22 6.37 35 36 12 5.90 11 5.41 37 7 2.31 51.65 38 39 40 10 5.84 41 5 1.89 13 4.92 42 10 4.03 13 5.24 43 23 5 8.291.40 25 7 9.01 1.97 44 8 2.51 4 1.26 45 5 1.60 17 7 4.29 2.24 46 15 7.2547 7 5 2.12 1.82 3 11 0.91 4.00 48 11 3.84 19 6.64 49 3 14 0.88 5.31 213 6.18 1.14 50 7 2.64 X 10.7 6.7 3.7 2.5 18.4 11.1 6.2 3.7 SD 9.0 2.82.7 1.5 13.9 8.4 4.6 3.0

Example 6 Analysis of Plasma Before and after Fasting

Table 5 depicts the results of the ELISA analysis of plasma collected in20 subjects before (fasting) and after (non-fasting). PROT is protein inmg/dl, LEP is leptin, LEP/PR is leptin/protein, AG is agouti relatedprotein, AG/PR is agouti related protein/protein, INS is insulin, INS/PRis insulin/protein, X is mean values of 20 subjects, and SD is standarddeviation of result.

TABLE 5 Plasma BE- BE- BE- BE- FORE AFTER FORE AFTER BE- FORE AFTER BE-FORE AFTER BE- SUB- PROT PROT LEP LEP FORE AFTER AG AG FORE AFTER INSINS FORE AFTER JECT mg/ml mg/ml pg/ml pg/ml LEP/PR LEP/PR pg/ml pg/mlAG/PR AG/PR μIU/ml μIU/ml INS/PR INS/PR 1 7.8 35 4 55 7 2 7.2 38 5 3 7.737 5 36 5 4 8.6 61 7 46 5 5 7.9 10661 1349 30 4 3 0 6 6.5 5345 51 8 1 07 6.9 51 7 17 2 8 7.6 38 5 9 7.9 34 4 41 5 10 7.4 28 4 6 1 11 7.1 53 712 6.2 6.2 1793 1714 289 276 65 10 95 60 15 10 13 7.2 5075 705 25 3 4 114 7.2 70 10 24 3 15 6.6 34 5 16 7.2 16860 2342 55 8 44 6 17 7.9 168602134 33 4 1 0 18 7.3 15826 2168 37 5 7 1 19 7.3 37 5 18 2 20 6.6 58 9 X7 7 5843 11321 781 1730 42 45 6 6 32 28 5 4 SD 1 1 4484 7240 534 973 1414 3 2 38 21 6 3

Example 7 Analysis of Insulin Concentration in Nasal Mucus, Plasma, andSaliva

Specimens of nasal mucus from different subjects were collected andanalyzed using ELISA. Table 6 depicts detection and measurement of humaninsulin in nasal mucus as compared to insulin in blood plasma and salivaunder several physiological and pathological processes. In controlsubjects, in the fasting state, insulin concentrations were similar ineach biological fluid measured. In the non-fasting state, the nasalmucus concentrations were significantly lower than in plasma or saliva.In obese subjects and in diabetics, in the fasting state, insulinconcentrations were similar in plasma and nasal mucus but slightlyelevated in saliva. However, in the non-fasting state, insulinconcentrations increased in plasma and saliva in response to increasedcarbohydrate intake but in nasal mucus, insulin levels decreased.

TABLE 6 Insulin in plasma, saliva, and nasal mucus PLASMA NASAL AGEWEIGHT GLUCOSE PLASMA SALIVA MUCUS (years) (lbs) mg/ml μIU/ml μIU/mlμIU/ml SUBJECTS CONTROLS (56) 56 ± 2 174 ± 8 FASTING  88 ± 1 17.1 ± 3.718.9 ± 2.3 19.0 ± 2.2 NON-FASTING 174 ± 8  29.4 ± 4.4 22.6 ± 2.6  7.5 ±0.9 ^(c) SUBJECTS (11) OBESE 51 ± 6 259 ± 26 FASTING 110 ± 24 20.0 ± 6.2^(†) 34.1 ± 7.9 18.4 ± 6.5 NON-FASTING 259 ± 26 38.2 ± 11.7 36.3 ± 6.3 7.5 ± 2.6 ^(d b a) DIABETES 51 ± 6 243 ± 52 FASTING 142 ± 37 22.7 ±10.2 36.8 ± 9.0 21.0 ± 9.3 NON-FASTING 243 ± 42 39.5 ± 19.0 38.2 ± 9.6 7.6 ± 2.3 ^(b) ( ) Subject number ^(†) MEAN ± SEM ^(a) p < 0.01 withrespect to non-fasting saliva ^(b) p < 0.005 with respect to fastingsaliva ^(c) p < 0.001 with respect to fasting nasal mucus andnon-fasting plasma, saliva ^(d) p < 0.05 with respect to fasting plasma,non-fasting plasma ^(e) p < 0.02 with respect to fasting plasma

Example 8 Analysis of Insulin Receptor Concentration in Nasal Mucus,Plasma, and Saliva

Table 7 depicts the detection and measurement of human insulin receptorin nasal mucus as compared to the insulin receptor in plasma and salivaunder several physiological and pathological conditions. In controlsubjects in the fasting state, insulin receptor concentrations measuredwere similar in plasma, saliva as well as nasal mucus. However, in thenon-fasting state, where there was little change in receptorconcentrations in plasma or saliva, there was a significant decrease innasal mucus receptor concentration. In obese subjects and in diabetics,in the fasting state, insulin receptor concentrations decreased in eachbiological fluid measured. However, in the non-fasting state, there wereno further decreases in receptor concentrations in plasma or in salivabut there was a decrease in receptor concentration in nasal mucusassociated with an increase in carbohydrate intake.

TABLE 7 Insulin receptor concentration in plasma, saliva, and nasalmucus PLASMA NASAL AGE WEIGHT GLUCOSE PLASMA SALIVA MUCUS (years) (lbs)mg/ml mg/ml mg/ml mg/ml SUBJECTS (56) CONTROLS (56) 56 ± 2 ^(†) 174 ± 8 84 ± 1 ^($) FASTING  88 ± 6 8.7 ± 1.8 ^(†) 7.6 ± 0.7 8.5 ± 1.7NON-FASTING 174 ± 8 7.5 ± 1.4 7.6 ± 0.6 3.2 ± 0.6 ^(a) SUBJECTS (11)OBESE 51 ± 6 ^(‡) 259 ± 26 FASTING 110 ± 24 ^($) 2.9 ± 0.9 ^(† b) 6.0 ±1.2 4.8 ± 0.6 ^(d) NON-FASTING 259 ± 26 3.0 ± 0.3 ^(b) 5.2 ± 0.8 ^(c)2.1 ± 0.5 THIN 122 ± 6 FASTING 8.4 ± 2.7 NON-FASTING 9.4 ± 4.6 DIABETES(4) 243 ± 52 FASTING 142 ± 37 4.0 ± 1.7 ^(b) 4.0 ± 0.6 ^(c) 4.7 ± 1.9NON-FASTING 243 ± 42 3.4 ± 0.6 ^(a) 4.6 ± 1.9 2.3 ± 1.6 ^(†) MEAN ± SEM^(‡) years ^($) mg/dl ^(a) p < 0.01 with respect to nasal mucus fasting,plasma (fasting, non-fasting), saliva (fasting, non-fasting) ^(b) p <0.005 with respect to controls ^(c) p < 0.001 with respect to controls^(d) p < 0.05 with respect to controls

These results in Tables 6 and 7 indicate that the characteristics ofnasal mucus reflect physiological and pathological conditions. Thedetection of the presence of insulin or insulin receptors in nasal mucusmay offer an alternative method for diagnosis of diabetes, otherdisorders of carbohydrate metabolism and physiological measurements ofinsulin and insulin receptors. Its ease of measurement using anon-invasive technique may be preferable to invasive techniques such asvenipucture. Its presence in nasal mucus may also offer a view intoother aspects of both human physiology and pathology. In the non-fastingstate insulin receptor concentration is down regulated to some extentunder some conditions but is uniformly regulated in nasal mucusindicating that, its concentration in nasal mucus may indicatephysiological phenomenon such as, appetite and brain, and the signalcharacteristics reflecting base human physiology and pathology. Itspresence may influence human immune and autoimmune responses.

Example 9 Pearson-Product-Moment Correlations of Insulin with PlasmaGlucose and Insulin with Weight

Table 8 depicts Pearson-product-moment correlations of insulin withplasma glucose and insulin with weight. There was little positivecorrelation among controls in insulin in plasma or insulin in saliva, ineither the fasting or non-fasting state. However, in nasal mucus thiscorrelation was negative, indicating a down regulated direction. Thissignal in nasal mucus may reflect a control mechanism of appetite. Thus,in nasal mucus there may be substances which reflect both physiologicalparameters common or uncommon to blood or saliva which provides methodsto diagnose body physiological and pathological events.

TABLE 8 Correlation (pearson product moment (r)) between insulin andindependent variable (N = 60) INSULIN CONCENTRATION PLASMA (r) SALIVA(r) NASAL MUCUS (r) NON- NON- NON- FASTING FASTING FASTING FASTINGFASTING FASTING VARIABLE (CONTROLS) PLASMA 0.16 0.08 0.25 0.26 −0.20−0.14 GLUCOSE(mg/dl) WEIGHT(lbs) 0.04 0.24 0.26 0.38 −0.14 −0.18VARIABLE (OBESE) PLASMA 0.87 −0.01  0.33 0.39 −0.30 −0.37 GLUCOSE(mg/dl)WEIGHT(lbs) 0.62 0.72 0.08 0.65 −0.44 −0.55 VARIABLE (DIABETES) PLASMA 1.00 ^(a)  1.00 ^(a) 0.65 0.59 −0.26 −0.64 GLUCOSE(mg/dl) WEIGHT(lbs)1.00  0.99 ^(a) 0.69 0.69 −0.28 −0.66 ^(a) p < 0.01

Example 10 Pearson-Product-Moment Correlations of Insulin Receptor withPlasma Glucose and Insulin Receptor with Weight

Table 9 depicts relationships between insulin receptor concentrationwith plasma glucose and weight. There was little positive correlationamong controls amongst insulin receptors in plasma or insulin receptorsin saliva, in either the fasting or non-fasting state. However, in nasalmucus this correlation was negative, indicating a down regulateddirection. This signal in nasal mucus may reflect a control mechanism ofappetite. Thus, in nasal mucus there may be substances which reflectboth physiological parameters common or uncommon to blood or salivawhich may provide methods to diagnose body physiological andpathological events.

TABLE 9 Correlation (pearson product moment (r)) between insulinreceptor concentration and independent variable (n = 60) INSULINRECEPTOR CONCENTRATION PLASMA (r) SALIVA (r) NASAL MUCUS (r) NON- NON-NON- FASTING FASTING FASTING FASTING FASTING FASTING VARIABLE (CONTROLS)PLASMA 0.20 0.12 −0.24 −0.14 0.03 0.11 GLUCOSE(mg/dl) WEIGHT(lbs) −0.11−0.07 −0.08 −0.19 −0.21 −0.17 VARIABLE (OBESE) PLASMA 0.70 0.20 −0.51−0.08 0.41 0.19 GLUCOSE(mg/dl) WEIGHT(lbs) −0.18 −0.33 0.09 −0.16 0.24−0.09 VARIABLE (DIABETES) PLASMA −0.57 −0.43 −0.78 0.25 0.15 −0.22GLUCOSE(mg/dl) WEIGHT(lbs) −0.60 −0.52 0.56 −0.34 0.18 −0.19

Example 11 Analysis of Caspase 3 in Nasal Mucus and Saliva

Caspase 3 is one of the apoptotic substances involved in the apoptoticprocess. Table 10 illustrates a comparison between the detection andmeasurement of caspase 3 in nasal mucus and in saliva in 18 subjects.The presence of caspase in nasal mucus is about 13% of that in salivaand reflects the magnitude of the apoptotic process. The presence ofcaspase in nasal mucus indicates the activity of cellular death in bothphysiological and pathological processes.

TABLE 10 Caspase 3 in human saliva and nasal mucus SALIVA NASAL MUCUSCASPASE 3 PROTEIN CASPASE 3 CASPASE 3 PROTEIN CASPASE 3 Subjects μg/mlmg/dl PROTEIN μg/ml mg/dl PROTEIN 18 2.87 ± 0.70^(†) 2.7 ± 0.2 2.88 ±0.28 0.38 ± 0.13 2.2 ± 0.2 0.38 ± 0.17 ^(†)MEAN ± SEM

Example 12 Analysis of TNFα in Nasal Mucus and Saliva

Table 11 illustrates detection and measurement of TNFα in nasal mucusand saliva in 75 subjects. Results indicate that TNFα in nasal mucus isabout 30 times higher than in saliva. Elevated levels of this substancein nasal mucus in diverse disease processes makes their diagnosispossible on a clinical basis since obtaining cellular diagnosis throughtissue biopsy can not only be invasive but also difficult and at timesdangerous. The concentration of TNFα in nasal mucus can be reflective ofunderlying disease processes and is easier to obtain. These results maymake the use of this fluid an important method of diagnosis for thesepathological processes which cannot be as conveniently made in plasma.These data suggest that various cancers can be diagnosed by measurementsof TNFα in nasal mucus and their treatment can be monitored by followingits concentration in nasal mucus. Since levels of TNFα may also reflectthe inflammatory aspects of disease processes inducing it, use of antiTNFα drugs through nasal administration may reflect a method of treatingthese various disease processes. Concentrations of TNFα in nasal mucusin patients with smell loss can be greater than for example, 5000 timesthat found in normal subjects thereby reflecting its function as a“death protein” indicator of excessive apoptosis as in its role incancer.

TABLE 11 TNFα in human saliva and nasal mucus SALIVA NASAL MUCUS TNFαPROTEIN TNFα TNFα PROTEIN TNFα Subjects μg/ml mg/dl PROTEIN μg/ml mg/dlPROTEIN 75 0.43 ± 0.03^(†) 3.1 ± 0.1 0.15 ± 0.01 12.8 ± 1.6^(a) 2.4 ±0.09^(a) 5.5 ± 1.3^(a) ^(†)MEAN ± SEM With respect to saliva ^(a)p <0.001

Example 13 Analysis of TNFR I in Nasal Mucus and Saliva

Table 12 illustrates detection and measurement of TNFR I in 47 subjects.Results indicate that TNFR I in nasal mucus is about 16 times itsconcentration in saliva and its concentration is significantly increasedover that found in plasma, red blood cells, or urine. The results showthat TNFR I present in nasal mucus can reflect activity of manyinflammatory, oncological and other pathological processes, includingtaste and smell dysfunction. Thus the methods of the present inventioncan be used to detect and establish clinical diagnoses of excessiveapoptosis and can be used as a treatment modality in inhibitingpathological apoptosis.

TABLE 12 TNF receptor I in human saliva and nasal mucus SALIVA NASALMUCUS TNFR I PROTEIN TNFR I TNFR I PROTEIN TNFR I Subjects μg/ml mg/dlPROTEIN μg/ml mg/dl PROTEIN 47 110 ± 13^(†) 3.1 ± 0.1 38 ± 5 1753 ±357^(a) 2.2 ± 0.1^(a) 837 ± 161^(a) ^(†)MEAN ± SEM With respect tosaliva ^(a)p < 0.001

Example 14 Analysis of TNFR II in Nasal Mucus and Saliva

Table 13 illustrates detection and measurement of TNFR II in 47subjects. Results indicate that TNFR II in nasal mucus is about 24 timesits concentration in saliva and its concentration in nasal mucus issignificantly higher than found in plasma, red blood cells, or urine.The results reflect that TNFR II present in nasal mucus provides a noninvasive method of diagnosing various pathological processes. Thus themethods of the present invention can be used to detect and establishclinical diagnoses of excessive apoptosis and can be used as a treatmentmodality in inhibiting pathological apoptosis.

TABLE 13 TNF receptor II in human saliva and nasal mucus SALIVA NASALMUCUS TNFR II PROTEIN TNFR II TNFR II PROTEIN TNFR II Subjects μg/mlmg/dl PROTEIN μg/ml mg/dl PROTEIN 47 48 ± 2^(†) 3.1 ± 0.1 17 ± 0.9 1126± 217^(a) 2.2 ± 0.1^(a) 578 ± 128^(a) ^(†)MEAN ± SEM With respect tosaliva ^(a)p < 0.001

Example 15 Analysis of TRAIL in Nasal Mucus and Saliva

Tables 14 and 15 illustrate detection and measurement of TRAIL in salivaand nasal mucus in normal subjects and in patients with smell loss. InTable 14, results indicate that TRAIL in nasal mucus is about 5 timeshigher than in saliva and both are significantly higher than in blood,red blood cells, or urine. TRAIL in nasal mucus in some patients withsmell and taste loss varies from 500-10,000 times higher than in normalsubjects and it may be elevated in nasal mucus in patients withinflammatory and oncological disease processes. Mean levels of increasedTRAIL in nasal mucus reveal levels significantly greater than in otherfluids. The results provide a non-invasive method for detecting TRAIL innasal mucus.

TABLE 14 Trail in saliva and nasal mucus in normal subjects and inpatients with smell loss SALIVA NASAL MUCUS TRAIL PROTEIN TRAIL TRAILPROTEIN TRAIL SUBJECTS (Number) μg/ml mg/dl PROTEIN μg/ml mg/dl PROTEINNORMALS(17)  336 ± 48^(†) 2.7 ± 0.1 122 ± 14 1639 ± 89  2.2 ± 0.01 770 ±35 PATIENTS(10) 353 ± 31 2.7 ± 0.2 141 ± 20  2584 ± 430 1.4 ± 0.2  4753± 1400 ^(†)MEAN ± SEM

TABLE 15 Trail in saliva and nasal mucus in normal subjects and inpatients with Hyposmia SALIVA FLOW TRAIL NASAL MUCUS PROTEIN TRAIL RATEFLOW PROTEIN TRAIL TRAIL SUBJECTS(Number) mg/dl μg/ml ml/sec RATE mg/dlμg/ml PROTEIN NORMALS(28)  2.29 ± 0.10^(†) 123 ± 6 0.80 ± 0.03 343 ± 132.65 ± 0.12 1990 ± 119 7.56 ± 28 PATIENTS(65) 2.96 ± 0.09  330 ± 25^(a)0.63 ± 0.03  582 ± 55^(a) 1.97 ± 0.11 4121 ± 54^(a)  3095 ± 591^(a) ()Subject number ^(†)Mean ± SEM ^(a)p < 0.001 with respect to normal

Example 16 Analysis of TRAIL in Nasal Mucus in Patients with HyposmiaBefore and after Treatment with Theophylline

Table 16 illustrates detection and measurement of TRAIL in nasal mucusin patients with hyposmia before and after treatment with theophyllineat various doses. Data indicate that treatment with theophylline whichreturned smell function to normal in a dose-dependent manner wasassociated with a dose-dependent decrease in TRAIL, which indicates adecrease in the abnormal apoptotic processes. These data indicate both abiochemical and functional improvement in smell function by treatmentwith theophylline.

TABLE 16 Nasal mucus Trail in patients with hyposmia before and aftertreatment with theophylline at various doses THEOPHYLLINE TREATMENTPRETREATMENT 200 mg 400 mg 600 mg TRAIL PROTEIN TRAIL PROTEIN TRAILPROTEIN TRAIL PROTEIN SUBJECTS (Number) μg/ml mg/dl μg/ml mg/dl μg/mlmg/dl μg/ml mg/dl PATIENTS(5) 2584 1.36 855 1.29 791 2.06 247 2.00NORMALS(9) 335 ^(†) MEAN ± SEM

Example 17 Analysis of IL2 in Saliva and Nasal Mucus

Table 17 illustrates levels of IL2 in human nasal mucus and saliva. IL2was not present in nasal mucus although it was found in plasma.

TABLE 17 IL 2 in human saliva and nasal mucus SALIVA NASAL MUCUS IL 2PROTEIN IL 2 IL 2 PROTEIN IL 2 SUBJECTS (Number) μg/ml mg/dl PROTEINμg/ml mg/dl PROTEIN (10) 0 3.1 ± 0.2^(†) 0 0 2.2 ± 0.1 0 ^(†)MEAN ± SEM

Example 18 Analysis of IL3 in Saliva and Nasal Mucus

Table 18 illustrates measured IL 3 in both human saliva and nasal mucus.Levels of IL 3 in nasal mucus were found to be about ½ the concentrationin saliva but both levels were higher than that found in plasma, redblood cells, or urine. IL 3 present in nasal mucus provides a noninvasive method of diagnosing various IL3 related diseases.

TABLE 18 IL 3 in human saliva and nasal mucus SALIVA NASAL MUCUS IL 3PROTEIN IL 3 IL 3 PROTEIN IL 3 SUBJECTS (Number) μg/ml mg/dl PROTEINμg/ml mg/dl PROTEIN (17) 140 ± 32^(†) 3.1 ± 0.2 48 ± 15 63 ± 24 2.2 ±0.1 43 ± 12 ^(†)MEAN ± SEM

Example 19 Analysis of Endostatin in Human Plasma, Urine, Saliva andNasal Mucus

Table 19 illustrates detection and measurement of endostatin in plasma,urine, saliva and nasal mucus in 15 subjects. Endostatin levels in nasalmucus were 5 times higher than in saliva and about 7% of that found inplasma. On the basis of the endostatin/protein ratio, levels in nasalmucus are about 14% of that found in plasma. Presence of this 20 KDprotein in nasal mucus illustrates a non-invasive method of detection ofendostatin in nasal mucus and its use in diagnosing various endostatinrelated diseases. It may also be indicative of its synthesis in nasalmucus.

TABLE 19 Endostatin in human plasma, urine, saliva and nasal mucusBIOLOGICAL ENDOSTATIN PROTEIN ENDOSTATIN FLUIDS (15) μg/ml mg/dl PROTEINPLASMA 94 ± 10^(†)  6.9 ± 0.10 15 ± 1    URINE  0.5 ± 0.04^(a) — —SALIVA 1.3 ± 0.3^(a) 2.6 ± 0.2 0.59 ± 0.04^(a) NASAL MUCUS 6.6 ± 1.3^(a)3.0 ± 0.2  2.0 ± 0.43^(a) ( ) Subject number ^(†)MEAN ± SEM ^(a)p <0.001 with respect to plasma

Example 20 Analysis of Erythropoetin (EPO) in Plasma, Urine, Saliva andNasal Mucus

Table 20 illustrates detection and measurement of EPO in plasma, urine,saliva and nasal mucus. EPO was not found in urine or saliva. The levelof EPO in nasal mucus was found to be between 1.1 and 4.5 times higherthan in plasma. Presence of EPO in nasal mucus illustrates anon-invasive method of detection of EPO in nasal mucus and its use indiagnosing various EPO related diseases.

TABLE 20 EPO in plasma, urine, saliva and nasal mucus ERYTHRO- ERYTHRO-BIOLOGICAL POETIN PROTEIN POETIN FLUIDS (27) μIU/ml mg/dl PROTEIN PLASMA13 ± 2^(†) 7.2 ± 0.1   2 ± 0.3 URINE 0 0 0 SALIVA 0 0 0 NASAL MUCUS 15 ±5  2.9 ± 0.1^(a) 9 ± 2^(b ) ( ) Subject number ^(†)MEAN ± SEM ^(a)p <0.001 with respect to plasma ^(b)p < 0.005 with respect to plasma

Example 21 Analysis of Bone Morphogenic Protein (BMP) in Plasma, Urine,Saliva and Nasal Mucus

Table 21 illustrates detection and measurement of BMP I in plasma,urine, saliva and nasal mucus in 20 subjects. BMP I was found in plasmabut not in urine, saliva or nasal mucus.

TABLE 21 BMP in plasma, urine, saliva and nasal mucus BIOLOGICAL BMPPROTEIN BMP FLUIDS (20) μg/ml mg/dl PROTEIN PLASMA 30 ± 6^(†) 3.6 ± 0.50 URINE 0 0 0 SALIVA 0 0 0 NASAL MUCUS 0 0 0 ( ) Subject number ^(†)MEAN± SEM

Example 22 Analysis of Brain Derived Neurotrophic Factor (BDNF) in HumanPlasma, Urine, Saliva and Nasal Mucus

Table 22 illustrates detection and measurement of BDNF in plasma, urine,saliva and nasal mucus in 20 subjects. BDNF was found in plasma andnasal mucus but not in urine or saliva. Levels of BDNF in plasma werehigher than in nasal mucus. The results indicate that nasal mucus may bea repository of the family of nerve growth factors and the concentrationof BDNF as shown in Table 22, may help understand both the physiologyand pathology of neurotrophic factors related to growth and homeostasisof cells in the nasal cavity as well as reporting on the presence ofthese factors in the systemic circulation.

TABLE 22 BDNF in human plasma, urine, saliva and nasal mucus BIOLOGICALBDNF PROTEIN BDNF FLUIDS (20) μg/ml mg/dl PROTEIN PLASMA 3391 ± 530  7.1± 0.01 447 ± 74  URINE 0 — SALIVA 0 2.7 ± 0.2 NASAL MUCUS 11 ± 7 3.2 ±0.3 8 ± 6 ( ) Subject number ^(†)MEAN ± SEM

Example 23 Analysis of Ciliary Neurotrophic Factor (CNTF) in HumanPlasma, Urine, Saliva and Nasal Mucus

Table 23 illustrates detection and measurement of CNTF in plasma, urine,saliva and nasal mucus in 19 subjects. Levels of CNTF in plasma andnasal mucus were found to be similar but lower in saliva. These resultsindicate that measurement of CNTF in nasal mucus may be used as an indexfor the levels of CNTF in plasma. The results provide a non-invasivemethod for the detection of CNTF in nasal mucus. The detection of CNTFin nasal mucus provides a non invasive method of diagnosing various CNTFrelated diseases.

TABLE 23 CNTF in human plasma, urine, saliva and nasal mucus BIOLOGICALCNTF PROTEIN CNTF FLUIDS (19) μg/ml mg/dl PROTEIN PLASMA 0.004 ± 0.0013.1 ± 0.1 0 URINE 0 SALIVA 0.001 ± 0.001 3.0 ± 0.1 0 NASAL MUCUS 0.003 ±0.001 2.2 ± 0.1 0 ( ) Subject number

Example 24 Analysis of Granulocyte Macrophage Growth Factor (GM-CSF) inHuman Plasma, Urine, Saliva and Nasal Mucus

Table 24 illustrates detection and measurement of GM-CSF in plasma,urine, saliva and nasal mucus in 16 subjects. Levels in nasal mucus werefound to be over 6 times that found in plasma. The results provide anon-invasive method for the detection of GM-CSF in nasal mucus. Theresults also indicate nasal mucus to be a source of GM-CSF. Thedetection GM-CSF present in nasal mucus provides a non invasive methodof diagnosing various GM-CSF related diseases.

TABLE 24 GM-CSF in human plasma, urine, saliva and nasal mucusBIOLOGICAL GM-CSF PROTEIN GM-CSF FLUIDS (16) μg/dl mg/dl PROTEIN PLASMA0.42 ± 0.31^(†) 7.2 ± 0.1 0.036 ± 0.034 URINE 0 0 SALIVA 0 0 NASAL MUCUS2.55 ± 0.8   2.9 ± 0.1   0.58 ± 0.25^(a) ( ) Subject number ^(†)MEAN ±SEM ^(a)p < 0.001 with respect to plasma

Example 25 Analysis of Hepatocyte Growth Factor (HGF) in Human Plasma,Urine, Saliva, and Nasal Mucus

Table 25 illustrates detection and measurement of HGF in plasma, urine,saliva and nasal mucus in 17 subjects. Concentrations of HGF in nasalmucus were found to be higher than that found in either plasma or urine.These results suggest that HGF may be synthesized in the serous glandsof the nose for a specific mechanism involved with nasal homeostasis aswell as a mechanism involved with systemic cell migration. The resultsprovide a non-invasive method for the detection of HGF in nasal mucus.

TABLE 25 HGF in human plasma, urine, saliva and nasal mucus BIOLOGICALHGF PROTEIN HGF FLUIDS (17) μg/ml mg/dl PROTEIN PLASMA 709 ± 91^(† ) 3.1± 0.1 100 ± 13    URINE 623 ± 126 — — SALIVA 0 0 NASAL MUCUS 2015 ±431^(a ) 2.2 ± 0.1 924 ± 227^(a) ( ) Subject number ^(†)MEAN ± SEM Withrespect to plasma and urine ^(a)p < 0.001

Example 26 Analysis of Platelet Derived Growth Factor (PDGF) in HumanPlasma, Urine, Saliva and Nasal Mucus

Table 26 illustrates detection and measurement of PDGF in human plasma,urine, saliva and nasal mucus in 18 subjects. Concentrations of PDGFexpressed per mg protein were found to be higher in saliva and nasalmucus than in plasma. These results suggest that PDGF may be synthesizedin the serous glands of the nose for a specific mechanism involved withnasal homeostasis. The results provide a non-invasive method for thedetection of PDGF in nasal mucus.

TABLE 26 PDGF in human plasma, urine, saliva and nasal mucus BIOLOGICALPDGF PROTEIN PDGF FLUIDS (18) μg/dl mg/dl PROTEIN PLASMA  510 ± 153^(†)6.9 ± 0.1 71 ± 21 URINE 5 ± 2 — — SALIVA 600 ± 176 2.6 ± 0.2 215 ±18^(a ) NASAL MUCUS 482 ± 87  3.0 ± 0.2 175 ± 32^(b ) ( ) Subject number^(†)MEAN ± SEM ^(a)p < 0.001 with respect to plasma ^(b)p < 0.02 withrespect to plasma

Example 27 Analysis of Carbonic Anhydrase VI (CA VI) Concentration

Table 27 illustrates decrease in CA VI in patients with smell and tasteloss. These results provide a method for the detection and measurementof CA VI in nasal mucus as an index of smell and taste loss and itscontinual measurement during treatment of these disorders in order tomonitor efficacy of therapy.

TABLE 27 CA VI concentrations in nasal mucus in normal subjects and inpatients with smell loss PROTEIN ZINC COPPER CA VI SUBJECTS mg/dl μg/Lμg/L mg/ml NORMALS (8)  3.41 ± 0.02^(†) 97 ± 8 102 ± 8  0.287 ± 0.056MEN (5) 2.96 ± 0.30 100 ± 11  78 ± 26 0.238 ± 0.03  WOMEN (3) 3.54 ±2.04 90 ± 8 103 ± 15 0.369 ± 0.26  PATIENTS (70)  2.27 ± 0.04 0.157 ±0.13  MEN (39)   2.21 ± 0.14^(a) 182 ± 17 118 ± 14 0.158 ± 0.020 WOMEN(31)   2.34 ± 0.18^(a) 171 ± 21 126 ± 12 0.155 ± 0.014 ( ) Subjectnumber ^(†)Mean ± SEM Compared to normals ^(a)p < 0.001

Example 28 Analysis of Loss of Smell Function by Disease Etiology

Table 28 illustrates loss of smell function by disease etiology withrespect to measurements of CA VI concentration in nasal mucus. Resultsindicate that patients with post influenza hyposmia hypogeusia (PIHH),allergic rhinitis and post anesthesia have significantly decreased CA VIconcentrations in nasal mucus. These results provide a method for thedetection and measurement of CA VI in nasal mucus as an index of smelland taste loss and its continual measurement during treatment of thesedisorders in order to monitor efficacy of therapy. The detection of CAVI in nasal mucus provides a non invasive method of diagnosing variousdiseases related to human physiology and pathology.

TABLE 28 Carbonic Anhydrase VI concentrations in nasal mucus in normalsubjects and in patients with smell loss PROTEIN ZINC COPPER CA VICONDITION mg/dl μg/L μg/L μg/ml NORMALS (11)  3.17 ± 0.48^(†) 97 ± 7 102± 7  0.287 ± 0.044 PIHH (26) 2.39 ± 0.19  139 ± 18^(a)  105 ± 11^(a)0.186 ± 0.02^(c ) ALLERGIC RHINITIS (25) 2.34 ± 0.19  234 ± 24^(a) 139 ±20  0.141 ± 0.018^(b) POST ANESTHESIA  (6)  1.65 ± 0.30^(b) 189 ± 60 139± 40  0.156 ± 0.047^(c) PHANTAGEUSIA  (5) 2.30 ± 0.59 170 ± 49 158 ± 310.180 ± 0.054 ( ) Subject number ^(†)Mean ± SEM Compared to normals^(a)p < 0.001 ^(b)p < 0.025 ^(c)p < 0.05

Example 29 Analysis of cAMP and cGMP in Human Nasal Mucus and in ParotidSaliva

Table 29 illustrates detection and measurement of cAMP and cGMP insaliva and in nasal mucus in normal subjects. Results show that patientswith smell loss had decreased levels of cAMP in their nasal mucus. Theseresults indicate that cAMP in nasal mucus can be an index of smell lossand that its secretion may be inhibited in smell loss. Thus, monitoringof cAMP in the nasal mucus can be a diagnostic tool for the treatment ofdiseases related to cAMP. Results also indicate that there was lesssignificant difference between cGMP in human nasal mucus and in parotidsaliva. The results provide a non-invasive method for the detection ofcAMP and cGMP in nasal mucus.

TABLE 29 Comparison of cAMP and cGMP in human nasal mucus and in parotidsaliva NASAL MUCUS PAROTID SALIVA^(††) TOTAL cAMP*  0.22 ± 0.07^(†$)2.00 ± 0.31 cGMP 0.25 ± 0.08 0.21 ± 0.04 MEN cAMP  0.21 ± 0.13^($) 1.58± 0.43 cGMP 0.28 ± 0.16 0.23 ± 0.06 WOMEN cAMP  0.23 ± 0.06^($) 3.38 ±0.35 cGMP 0.24 ± 0.13 0.20 ± 0.07 PROTEIN** TOTAL 3.24 ± 0.22 3.11 ±0.18 MEN 3.32 ± 0.02 3.39 ± 0.34 WOMAN 3.51 ± 0.75 2.93 ± 0.02 *inpmol/ml **mg/ml ^(†)MEAN ± SEM ^(††)Collected in 171 subjects ^($)p <0.001 compared to parotid saliva

Example 30 Analysis of cAMP and cGMP in Human Nasal Mucus in NormalSubjects and in Patients

Table 30 illustrates comparison of the measurement of cAMP and cGMP innormal subjects with the patients with taste and smell loss. Resultsshow that patients with smell loss had decreased levels of cAMP in theirnasal mucus. These results indicate that cAMP in nasal mucus can be anindex of smell loss and that its secretion may be inhibited in smellloss. Thus, monitoring of cAMP in the nasal mucus can be a diagnostictool for the treatment of diseases related to cAMP. Results alsoindicate that there was less significant difference between cGMP innasal mucus in normal subjects or in patients with hyposmia.

TABLE 30 Comparison of cAMP and cGMP in human nasal mucus in normalsubjects and in patients with smell loss (Hyposmia) cAMP cGMP pmol/mgpmol/mg PROTEIN pmol/ml protein pmol/ml protein mg/min CONDITION NORMAL(41)  0.22 ± 0.02^(†) 0.08 ± 0.01 0.25 ± 0.04 0.06 ± 0.01 3.37 ± 0.19MEN (34) 0.21 ± 0.01 0.05 ± 0.01 0.28 ± 0.02  0.04 ± 0.004 3.32 ± 0.13WOMEN  (7) 0.23 ± 0.06 0.10 ± 0.04 0.24 ± 0.13 0.10 ± 0.04 3.59 ± 1.02PATIENTS (146)   0.14 ± 0.02^(d) 0.07 ± 0.01 0.20 ± 0.02 0.12 ± 0.01 2.48 ± 0.08^(b) MEN (63)  0.14 ± 0.02^(c) 0.07 ± 0.02 0.25 ± 0.03 0.12± 0.02  2.50 ± 0.13^(b) WOMEN (83) 0.15 ± 0.02 0.07 ± 0.01 0.17 ± 0.020.11 ± 0.01  2.46 ± 0.11^(c) ( ) Subject number ^(†)Mean ± SEM ^(b)p <0.005 compared to normals ^(c)p < 0.01 compared to normals ^(d)p < 0.05compared to normals

Example 31 Analysis of cAMP and cGMP Concentrations in Nasal Mucus ofPatients

Table 31 illustrates detection and measurement of cAMP and cGMPsecretion in nasal mucus in patients with graded severity of smell loss(anosmia<Type I hyposmia<Type II hyposmia from most severe to leastsevere). Data indicates that as degree of smell loss increased, levelsof cAMP in nasal mucus decreased. These data confirm the relationshipbetween cAMP secretion in nasal mucus and degree of smell loss. Resultsalso indicate that there was less significant difference between cGMP innasal mucus in normal subjects or in patients with hyposmia.

TABLE 31 cAMP and cGMP concentrations in nasal mucus in patients withvarious degrees of smell loss TOTAL cAMP cGMP PROTEIN cAMP PROTEIN cGMPPROTEIN PATIENTS mg/ml pmol/ml pmol/mg pmol/ml pmol/mg ANOSMIA  (2) 1.410.004 0.003 0.179 0.127 HYPOSMIA TYPE I (17) 2.61 ± 0.29  0.116 ± 0.04*0.034 ± 0.01 0.225 ± 0.05 0.101 ± 0.02 TYPE II (64) 2.56 ± 0.13 0.193 ±0.03  0.102 ± 0.02^(a) 0.189 ± 0.03 0.079 ± 0.01 TYPE III NORMALSUBJECTS (10) 3.70 ± 0.67 0.225 ± 0.67 0.088 ± 0.04 0.356 ± 0.13 0.094 ±0.03 ( ) Patient number *Mean ± SEM ^(a)p < 0.001 with respect to Type Ihyposmia

Example 32 Analysis of Nitric Oxide (NO) in Saliva and in Nasal Mucus

Table 32 illustrates detection and measurement of NO in human saliva andnasal mucus. NO was found to be present in both saliva and nasal mucusand its mean concentration in saliva were 21% lower in patients than innormal subjects whereas in nasal mucus mean levels were 25% lower inpatients. Treatment which increases cAMP in nasal mucus and improvessmell function may be mirrored by increases in nasal mucus NO. Thedetection of NO in nasal mucus provides a non invasive method ofdiagnosing various diseases related to human physiology and pathology.

TABLE 32 NO in saliva and nasal mucus in normal subjects and in patientswith smell loss SALIVA NASAL MUCUS NO PROTEIN NO/ NO PROTEIN NO/SUBJECTS μg/ml mg/dl PROTEIN μg/ml mg/dl PROTEIN NORMALS (15) 0.57 ±0.03^(†)  2.6 ± 0.1 0.23 ± 0.02 0.48 ± 0.08 2.3 ± 0.15 0.22 ± 0.05PATIENTS (34) 0.45 ± 0.06 2.8 ± 0.1 0.18 ± 0.03 0.36 ± 0.03 2.0 ± 0.080.21 ± 0.03 ( ) Subject number ^(†)MEAN ± SEM

Example 33 Analysis of Nitric Oxide (NO) in Nasal Mucus in PatientsBefore and after Theophylline Treatment

Table 33 illustrates NO levels in nasal mucus in patients treated withTheophylline in various doses before and after drug treatment. NO levelsin nasal mucus changed following the treatment of patients with smellloss. Results show treatment of patients with graded increasing doses oftheophylline and measurement of both smell function and NO in nasalmucus in patients with hyposmia. Results indicated that prior to thetreatment levels of NO in nasal mucus were lower than in normalsubjects. After treatment with theophylline in graded doses there wereincreases in nasal mucus NO associated with graded increases in smellfunction. These data demonstrate that treatment with drugs that increasesmell function to or toward normal, returns smell function to normal.These results also demonstrate the measurements of various substances innasal mucus as an index of both human physiology and pathology ofvarious diseases. Its continual measurement during treatment of thesedisorders helps in monitoring efficacy of therapy. The detection of NOin nasal mucus provides a non invasive method of diagnosing variousdiseases related to human physiology and pathology.

TABLE 33 NO in nasal mucus in patients with Theophylline in variousdoses before and after drug treatment PRETREATMENT 200 mg 400 mg 600 mgNO Protein NO Protein NO Protein NO Protein SUBJECTS μg/ml mg/dl μg/mlmg/dl μg/ml mg/dl μg/ml mg/dl Patients (12) 0.35 ± 0.07 1.6 ± 0.3 0.25 ±0.01 1.7 ± 0.3 0.40 ± 0.06 2.1 ± 0.10 0.59 ± 0.16 1.9 ± 0.15 Normal 0.61± 0.20 ( ) Subject number

Example 34 Analysis of Insulin Like Growth Factor (IGF 1) in HumanSaliva and Nasal Mucus

Table 34 illustrates detection and measurement of IGF 1 in human salivaand nasal mucus in 26 subjects. Results show that IGF 1 concentration innasal mucus was significantly greater than in saliva. Results indicatethat the measurement of nasal mucus IGF 1 can be used as an index ofhuman physiology and pathology. The detection of IGF 1 in nasal mucusprovides a non invasive method of diagnosing various diseases related tohuman physiology and pathology.

TABLE 34 IGF 1 in human saliva and nasal mucus SALIVA NASAL MUCUS IGF 1PROTEIN IGF 1/ IGF 1 PROTEIN IGF 1/ μg/ml mg/dl PROTEIN μg/ml mg/dlPROTEIN SUBJECTS (26) 11.4 ± 0.5^(†) 3.1 ± 0.2 4.6 ± 0.4 13.7 ± 0.4^(b)2.2 ± 0.2 8.9 ± 1.0^(a) ( ) Subject number ^(†)MEAN ± SEM With respectto saliva ^(b)p < 0.005 ^(a)p < 0.001

Example 35 Analysis of TNF∝, TNFR₁, and TNFR₂, in Nasal Mucus ofPatients

Table 35 illustrates detection and measurement of TNFα, and soluble TNFreceptors 1 and 2, moieties in the nasal mucus. TNFα, and soluble TNFreceptors 1 and 2 increase in systems undergoing excessive apoptosis.Treatment with 600 mg theophylline restored smell function to or towardnormal in these patients. There was a significant dose-response decreasein each moiety related to a stepwise increase in theophylline treatmentassociated and a stepwise improvement in olfactory acuity. These resultssuggest that pathological apoptosis of olfactory epithelial anatomycauses smell loss in patients with hyposmia; this process is reversedwith theophylline therapy which restores smell function in thesepatients. These results illustrate biochemical parameters associatedwith return of smell function in patients treated with theophylline.

TABLE 35 TNF∝, TNFR₁, TNFR₂, in nasal mucus in patients with hyposmiatreated with Theophylline Theophylline treatment (mg/l) PATIENTS PRE 200400 600 TNF∝* (24)   18.3 ± 6.1^(†) 20.0 ± 2.8  12.1 ± 2.1^(d)  7.4 ±1.8^(a) TNFR₁ (19) 2,353 ± 718 3,148 ± 663 1,146 ± 220^(b) 1,220 ±286^(ab) TNFR₂ (18) 1,747 ± 535 1,952 ± 339  949 ± 180^(c) 916 ± 344 *inpg/ml ( ) Subject Number ^(†)Mean ± SEM ^(a)p < 0.001 with respect to200 mg. ^(b)p < 0.001 with respect to 200 mg. ^(c)p < 0.025 with respectto 200 mg. ^(d)p < 0.05 with respect to 200 mg.

Example 36 Analysis of TNF∝ in Nasal Mucus in Patients Before and afterTreatment with Theophylline

These studies were extended to reveal levels of TNFα in nasal mucus ofpatients with graded loss of smell following treatment with theophylline(Table 36). Smell loss was graded such that loss was greatest in Type Ihyposmia, less in Type II and still less in Type III (I>II>III).Pretreatment levels of TNFα were significantly higher in patients withType I hyposmia than in Type II hyposmia consistent with their greaterdegree of smell loss. Following treatment which was effective inrestoring smell function toward normal, levels of TNFα decreased in eachpatient group consistent with each increased dose of theophylline whichgenerated a dose dependent increase in smell function—as theophyllinedose increased, smell function increased and TNFα levels decreased. InType I hyposmia TNFα decreased 40% after treatment with 400 mgtheophylline and 57% after treatment 600 mg. In Type II hyposmia TNFαdecreased 11% after treatment with 600 mg theophylline.

TABLE 36 TNF∝ in nasal mucus in patients with various types of hyposmiatreated with theophylline HYPOSMIA Theophylline treatment* TYPE PRE 200400 600 I (8) 26.0 ± 6.5^(tb) — 15.6 11.2 ± 4.4 II (13) 5.3 ± 1.0 14.9 ±2.5 11.5 ± 3.2   4.3 ± 0.8^(a) III (3) —   1.2 ± 0.7^(bc) *in mg orallydaily ( ) Subject Number ^(t)Mean ± SEM ^(a)p < 0.001 with respect to200 mg ^(b)p < 0.001 with respect to Type I (600 mg) ^(c)p < 0.01 withrespect to Type II (600 mg) ^(d)p < 0.001 with respect to Type II, pretreatment

Example 37 Analysis of TNFR 1 in Nasal Mucus in Patients Before andafter Treatment with Theophylline

TNF Receptor 1 (TNFR1) exhibited similar results in nasal mucus inpatients with smell loss after treatment with theophylline (Table 37).With increased smell loss pre treatment TNFR 1 levels were increased innasal mucus in patients with Type I hyposmia (whose smell loss wasincreased over that of patients with Type II hyposmia). Aftertheophylline treatment, as a dose of drug increased, TNFR 1 levelsdecreased in Type I associated with increases in smell function. Levelsof TNFR 1 at 600 mg theophylline were systematically decreased inrelation to degree of smell loss. The greater was the smell loss, thehigher was the level of TNFR 1.

TABLE 37 TNFR 1 in nasal mucus in patients with various types ofhyposmia treated with theophylline HYPOSMIA Theophylline treatment* TYPEPRE 200 400 600 I (7) 4,626 ± 1,647^(tb) 6,521 ± 1,304^(c) 1,498 1,832 ±704^(a )  II (10) 837 ± 60    1,462 ± 371   1,087 ± 335 862 ± 335 III(2) — — — 585 ± 335 *in mg orally daily ( ) Subject Number ^(t)Mean ±SEM ^(a)p < 0.005 with respect to 200 mg ^(b)p < 0.02 with respect toType II pre treatment ^(c)p < 0.01 with respect to Type II or 200 mg

Example 38 Analysis of TNFR2 in Nasal Mucus in Patients Before and afterTreatment with Theophylline

TNR Receptor 2 (TNFR 2) in nasal mucus exhibited similar results (Table38). Pretreatment with theophylline in patients with the most severesmell loss (Type I hyposmia) exhibited higher TNFR 2 levels in nasalmucus than did patients with less severe smell loss (Type I hyposmia).With a dose dependent increase of theophylline treatment levels of TNFR2 in nasal mucus decreased consistent with a dose dependent increase insmell function. Levels of TNFR 2 in patients with Type I and Type IIhyposmia decreased almost 50% on 600 mg theophylline consistent withtheir greatest return of smell function. At this highest level oftheophylline levels of TNFR 2 were decreased in relationship to thedecrease in smell function—TNFR 2, Type I>Type II>Type III; smell loss,Type I>Type II>Type III.

TABLE 38 TNFR 2 in nasal mucus in patients with various types ofhyposmia treated with theophylline HYPOSMIA Theophylline treatment* TYPEPRE 200 400 600 I (7) 2,718 ± 1,125^(t) 3,100 ± 1,184 1,491 ± 1,102 II(10) 1,145 ± 625  1,378 ± 480  1,014 ± 272 553 ± 158 III (2) — — — 436 ±443 *in mg orally daily ( ) Subject Number ^(t)Mean ± SEM ^(a)p < 0.001with respect to 200 mg ^(b)p < 0.001 with respect to Type I (600 mg)^(c)p < 0.01 with respect to Type II (600 mg)

Example 39 Analysis of Endoglin in Human Plasma, Urine, Saliva and NasalMucus

Table 39 illustrates detection and measurement of Endoglin in the nasalmucus. Results indicate that the measurement of nasal mucus Endoglin canbe used as an index of human physiology and pathology. The detection ofEndoglin in nasal mucus provides a non invasive method of diagnosingvarious diseases related to human physiology and pathology.

TABLE 39 Endoglin in human plasma, urine, saliva and nasal mucus PLASMAURINE SALIVA NASAL MUCUS ENDOGLIN PROTEIN ENDOGLIN ENDOGLIN ENDOGLINENDOGLIN PROTEIN ENDOGLIN SUBJECTS mg/ml mg/dl PROTEIN mg/ml mg/ml mg/mlmg/dl PROTEIN (33) 2.7 ± 0.1^(†a) 7.1 ± 0.1 0.38 ± 0.1 0 0 0.2 ± 0.1 2.8± 0.1 0.07 ± 0.01 ( ) Subject number ^(†)MEAN ± SEM ^(a)p < 0.001 withrespect to nasal mucus

Example 40 Increased Carbonic Anhydrase (CA) I, II and VI, Zinc andCopper after rTCMS

Ninety three patients, aged 18-85 y (52±2 y, Mean±SEM), 49 men, aged29-74 y (51±3 y) and 44 women, aged 20-85 y (53±3 y) with hyposmia,hypogeusia, and subsequent phantageusia (distortion of taste independentof any oral stimulus) and/or phantosmia (distortion of smell independentof any environmental odor) were studied before and after rTCMS in asingle blind placebo controlled fixed sequence clinical trial.

Patient symptoms persisted for 0.4-30 y (6.9±1.5 y) prior to rTCMS.Physical examination of head and neck including examination of oral andnasal cavities (the nose examined by anterior rhinoscopy with use ofvasoconstrictor agents) was within normal limits. Neither neurologicalnor psychiatric abnormalities other than taste and/or smell dysfunctionwas present in any patient. Anatomical magnetic resonance imaging (MRI)of brain and electroencephalographic (EEG) studies in all patients werewithin normal limits.

rTCMS was performed with a Cadwell magnetic pulse stimulator (Kennewick,Wash.) monitored with a TECA TD20 (Pleasantville, N.Y.) wave formgenerator, as described in Cicinelli, P., et al., Eletroenceph. Clin.Neurophys, 1997, 105:438-450; Henkin, Robert, et al., FASEB J., 2002,16:A878; Henkin, R. I., Encyclopedia of Neuroscience (3^(rd) Ed),Adelman, G. Smith, B H eds, Birkhauser, Boston, 2003, and; Moharram, R.,et al., FASEB J., 2004, 18:A201, all incorporated by reference in theirentirety herein.

Stimulation was applied in a fixed manner to four skull locations (leftand right temporoparietal, occipital, frontal of patients). Stimulusfrequency was 1 pulse given per 1-3 sec for 30-90 sec with 20 pulsesgiven at each location. Repeat stimulation was performed in all patientsin whom increased sensory acuity and/or decreased sensory distortionsoccurred; repetition continued (two-six applications) until no furtherincrease in sensory acuity and/or decrease in sensory distortionsoccurred.

One hour before and one to two hr after completion of rTCMS, venousblood and parotid saliva were collected. Whole venous blood was placedinto zinc free tubes containing 100 μl of zinc free heparin, on ice,centrifuged at 3000 rpm at 4° C., plasma removed and stored at −20° C.until assayed. Erythrocytes were washed and treated as described inAgarwal, R. P., et al., Bio. Tr. Elem. Res., 1985, 7:199-208,incorporated by reference in its entirety herein. Parotid saliva wascollected in four plastic tubes on ice using a modified Lashley cupapplied to Stensen's duct with maximal stimulation using reconstitutedlemon juice applied to the lingual surface as described in, Henkin, R.I., et al., Proc. Nat. Acad. Sci. USA, 1975, 72:488-492 and Henkin, R.I., et al., Amer. J. Med. Sci., 1976, 272:285-299, both incorporated byreference in their entirety herein. The first three tubes were collectedat two min intervals, the fourth tube until approximately eight ml wascollected. For convenience only results of saliva from the fourth tubewill be presented. Five hundred μl of saliva from the fourth tube wasplaced in dry ice immediately after collection and stored at −60° C.until measurements by SELDI-TOF mass analysis was performed.

Zinc and copper were measured in each tissue by atomic absorptionspectrophotometry (AAS) by using a double beam ThermoJarrell Ash video22 (Franklin, Mass.) AAS modified by the Maxwell Instrument Company(Salisbury, N.C.), the methods previously described in, Henkin, R. I.,et al., Amer. J. Med. Sci, 1999, 318, 380-391; Agarwal, R. P., et al.,Bio. Tr. Elem. Res., 1985, 7:199-208; Henkin, R. I., et al., Amer. J.Med. Sci., 1976, 272:285-299, and; Meret, S., et al., Clin. Chem., 1971,17:369-373, all incorporated by reference in their entirety herein.Saliva protein was determined by measurement of total peptide content byuse of absorbance at A215-A225 (called 4215) and the extinctioncoefficient. CA activity was measured by a modification of the method ofRichli, E. E., et al., J. Biol. Chem., 1964, 239:1065-1078, incorporatedherein by reference in its entirety. Saliva samples stored at −60° C.were thawed and 1 μl directly spotted on an H4 Protein Chip array (prewashed with 0.1% TFA in 50% aqueous acetonitrile) and their proteinprofile examined on a Ciphergen (Fremont, Calif.) PBS IIc mass analyzer.

Samples were first incubated in a humid chamber for 5-10 minutes at roomtemperature, then washed with 5% aqueous acetonitrile, dried and 1 μlmatrix added (sinapinic acid in 0.1% TFA, 50% aqueous acetonitrile).Samples were allowed to dry again and subjected to SELDI-TOF analysis onthe PBS IIc. Protein peaks were characterized by their apparentmolecular weight based on their mass/charge ratio (m/z).

Following initial observation of biochemical changes after rTCMSsubsequent measurements in all biological fluids were performed in ablinded manner; all samples were coded and results uncoded only afterall analyses were completed. Mean±SEM for each parameter was determinedfor each condition pre and post rTCMS. Differences between eachcondition were calculated for each parameter and significance ofdifferences determined by parametric (differences betweenundifferentiated means, paired t tests, X²) and non parametric (signtest) statistics.

Results:

After rTCMS mean CA VI activity and salivary zinc and copperconcentrations increased significantly as did mean CA I, II activity andplasma copper concentrations (Table 40). Significant increases in bothCA I, II and CA VI activity were also measured using paired comparisons(p<0.01 Student t test) and the sign test (p<0.05, Student t test) (datanot shown). These latter data are reflected in results shown in Table 41in which changes pre and post rTCMS are shown. Increased CA VI wasmeasured in 87% of patients with changes varying from −5% to +153% (meanchange, +17%) (Table 41); compared to chance changes of this magnitudewould occur <5 times in 1000 (X²). Increased CA I, II was measured in93% of patients with changes varying from −2% to +56% (mean change,+11%) (Table 41); compared to chance changes of this magnitude wouldalso occur <1 time in 1000 (X²). Increased plasma and erythrocyte zincand copper concentrations were measured in 91-93% of patients (Table41); compared to chance changes of this magnitude would also occur <1time in 1000 (X²).

TABLE 40 Changes in plasma, erythrocyte and saliva CA I, II and VI, zincand copper before (pre) and after (post) rTCMS in 93 patients rTCMSCONDITION PRE POST PLASMA Zn (μg/dl) 85 ± 2* 88 ± 2  Cu (μg/dl) 101 ± 2 109 ± 3^(a )  ERYTHROCYTES Zn (μg/gHb) 38.0 ± 0.5  39.7 ± 0.5^(b)  Cu(μg/gHb)  2.1 ± 0.04 2.2 ± 0.05 CA I, II (μg/g protein) 3.12 ± 0.06 3.45± 0.06^(d ) SALIVA Zn (μg/L) 103 ± 5  121 ± 5^(b)  Cu (μg/L) 13 ± 1  22± 3^(c )  CA VI (μg/g protein) 0.153 ± 0.009 0.197 ± 0.008^(d) *Mean ±SEM CA, carbonic anhydrase Compared to pre rTCMS ^(a)p < 0.05 ^(b)p <0.025 ^(c)p < 0.01 ^(d)p < 0.001

TABLE 41 Changes in plasma, erythrocyte and saliva carbonic anhydrase I,II and VI, zinc and copper before (pre) and after repetitivetranscranial magnetic stimulations (rtcms) in patients with decreasedsensory acuity and presence of sensory distortions rTCMS PRE vs. POSTNUMBER CONDITION INCREASED DECREASED UNCHANGED INCREASED % PLASMA Zn(μg/dl) 61 6 0 91 Cu (μg/dl) 62 5 0 93 ERYTHROCYTES Zn (μg/gHb) 66 1 099 Cu (μg/gHb) 62 4 1 93 CA I, II (μg/g 62 4 1 93 protein) SALIVA Zn(μg/L) 49 18 0 73 Cu ((μg/L) 51 16 0 76 CA VI (μg/g 60 5 2 90 protein)

SELDI-TOF mass spectrometry revealed a peak at m/z 21.5K in the postrTCMS spectra which was absent in the pre rTCMS spectra (FIG. 10). Alsopresent in the post rTCMS spectra was a repetitive protein patternseparated by intervals of approximately 5K m/z (FIG. 10). Similarpatterns were observed in about ⅓ of patients studied.

Thus, the present study illustrates that several salivary proteins canbe induced after rTCMS (FIG. 10). Increased enzyme activities and zincand copper concentrations usually persisted two-four wk after rTCMS;however, over time there was a slow, gradual decrease in enzymeactivities and in plasma, erythrocyte and saliva zinc and copperconcentrations as well as a loss of sensory acuity and subsequent returnof sensory distortions.

This study demonstrates that biochemical changes may occur after rTCMS.Since changes in taste and smell function occur in several neurologicaldisorders these results may also relate to other conditions such asepilepsy, Parkinsonism, Alzheimer disease, head injury, and motor neurondisease in which rTCMS can be an effective therapeutic agent.

Example 41 Recovery of Taste and Smell Function Following rTCMS

Seventeen right handed Caucasian patients, five men, aged 40-74 y (58±7y, X±SEM), 12 women, aged 30-76 y (51±5 y) were studied. Each had mildto severe persistent hyposmia and hypogeusia as well as mild to severepersistent birhinal phantosmia and/or global oral phantageusia; thesensory distortions were profound enough to interfere with normal lifepursuits. Acuity loss persisted for 6 mo to 30 y (4.1±2 y) and sensorydistortions persisted for 3 mo to 30 y (3.7±2 y) prior to clinic visit.Etiologies which initiated their symptoms were head injury (4 patients),post influenza-like infection (PIHH, 7 patients), idiopathic causes (4patients) and drug reactions (2 patients). Each of the 17 patients whopresented with these symptoms was treated with rTCMS.

None had any neurological symptom other than loss of sensory acuity andpresence of sensory distortions. None had any psychiatric symptom otherthan some depression associated with persistence of these cognitiveimpairments. Symptoms were unrelieved by any prior treatment withmultiple agents including antiepileptics, anxiolytics, antidepressants,trace metals, vitamins and a variety of alternative treatment modalitiesincluding herbal remedies, acupuncture, chiropractic techniques andhypnosis. Physical examination of each patient, including examination ofthe head and neck and general neurological examination, was withinnormal limits. Both anatomical brain MRI and electroencephalograms werewithin normal limits in each patient.

Measurement Techniques:

A battery of tests measuring taste and smell acuity and character anddegree of sensory distortions were administered to each patient. Tasteand smell acuity were determined by standard three stimuli forced choicestaircase techniques (Henkin R. I., Amer. J. Med. Sci. 1976,272:285-299, incorporated herein by reference in its entirety) by whichdetection (DT) and recognition (RT) thresholds and magnitude estimation(ME) for four tastants [NaCl (salt), sucrose (sweet), HCl (sour) andurea (bitter)] and four odorants [pyridine (dead fish), nitrobenzene(bitter almond), thiophene (old motor oil) and amyl acetate (bananaoil)] were determined, and reference values established for a largenumber of normal subjects. Results for DT and RT, in mmol/L and M/L fortastants and odorants, respectively, were converted into bottle units(BU) and compared to previously established standards. Magnitudeestimation (ME) was determined, calculated in % for each stimulus andcompared to previously described standards in Henkin et al.Otolaryngology, 1993, vol. 2, p 1-86 and Henkin et al. Drug safety,11:310-377, 1994, incorporated herein by reference in its entirety.

Sensory distortions were graded daily in intensity, duration andfrequency using a written record on a 0-100 scale for 3 d-4-wk prior torTCMS; 0 reflected total absence of sensory distortions, 100 reflectedthe digitized composite of the most intense distortion experienced overthe entire day. Records were reviewed prior to the study to insureadequate understanding of symptom grading. The entire battery ofcognitive measurements was obtained at the initial patient visit andrepeated immediately prior to and after each rTCMS treatment. Thisbattery was also repeated at variable intervals (1 day, 2-4-wk, 6-46 mo)after each rTCMS treatment. Each test battery was performed independentof knowledge of any prior test result.

Treatment Protocol:

rTCMS was performed with a Cadwell magnetic pulse stimulator (Kennewick,Wash.) monitored by a TECA TD20 (Pleasantville, N.Y.) wave formgenerator. Stimulation was applied by a single open quadrangular 12×12cm coil. Three sequential stimulation protocols were used. The first twowere considered placebo or sham trials.

For the initial sham trial, 20 stimuli at intervals of 1-3 sec wereapplied sequentially at the lateral acromial process of the clavicle(near Erb's point) (a) to the anterior right shoulder, then (b) anteriorleft shoulder at 20-30% maximal output [20-30% of 1.5T or ˜0.2-0.4 T(since stimulus delivery was non-linear)] and then (c) to the back ofthe mid neck region (at the level of C5-8 at 30-40% maximal output or˜0.4-0.8T); mild muscle group flexion of arm and hand muscles (shoulderstimulation) and neck, strap and facial muscles (neck) followed eachstimulation and was visually monitored.

For the second sham trial, 20 stimuli at intervals of 1-3 sec wereapplied sequentially to four skull regions (left temporoparietal,occipital, right temporoparietal, frontal) at 20% maximal output; thiswas considered subthreshold stimulation since no peripheral muscleresponses occurred.

For the treatment trial, 20 stimuli at intervals of 1-3 sec were appliedat 40-55% maximal output (˜0.8-LIT) to each skull location as in thesecond sham trial noted above. Muscle responses to this latterstimulation were present and monitored by visual observation (e.g.,right/left thenar and/or phalangeal flexion with left/righttemporoparietal stimulation, respectively).

After each 20 stimuli of sham or treatment stimulation at eachanatomical location, olfactory response to presentation of a single odor(one concentration of an odorant whose DT, RT and ME were previouslydetermined) and/or changes in intensity and character of phantageusiaand/or phantosmia (previously determined) was recorded. If any change inolfactory acuity or in sensory distortion occurred after anystimulation, stimulation at that location at that same intensity wasrepeated two-six times until no further change occurred.

Outcome Measures:

Mean±SEM of changes in taste and smell acuity (DT, RT, ME) and inintensity of sensory distortions before and after each rTCMS treatmentwere calculated and significance of differences determined by Student'st tests. Differences were also calculated using paired t tests withsignificance of differences pre and post rTCMS determined by Student's ttest.

Results: Pre rTCMS I

Taste:

Mean DTs and RTs for all tastants were above normal (i.e. acuity wasdecreased). Mean MEs for all tastants were below normal (i.e. acuity wasdecreased) (Table 42). Mean DT and RT for all tastants except DT for HClwere significantly above normal and mean MEs for all tastants weresignificantly below normal.

Smell:

Mean DTs and RTs for all odorants were above normal (i.e. acuity wasdecreased) and mean MEs for all odorants were below normal (i.e. acuitywas decreased) (Table 42). Mean DT and RT for all odorants (except DTsfor pyridine, thiophene and amyl acetate) and mean MEs of all odorantswere significantly above normal (Table 42).

Sensory Distortions:

Phantageusia intensity was 82±7%. Phantosmia intensity was 72±14% (Table43). There were no gender differences in either phantageusia orphantosmia intensity (Table 43).

Results: Post rTCMS I

Placebo or Sham Stimulation (0.2-0.4T):

No subjective or objective changes in either taste and/or smell acuityor in character or intensity of sensory distortions occurred in anypatient following stimulation of shoulders or neck or in any skulllocation.

Treatment Stimulation (0.8-1.1T):

Taste:

Mean DTs and RTs for all tastants decreased (i.e., acuity increased) andmean MEs for all tastants increased (i.e. acuity increased) (Table 42).Mean DT and RT returned to normal levels for NaCl, sucrose and HCl asdid DT for urea and ME for all tastants (Table 42). Only mean RT forurea did not return to normal although it was significantly lower thanbefore treatment (Table 42).

Smell:

Mean DTs and RTs for all odorants decreased (i.e., acuity increased) andmean MEs for all odorants increased (i.e., acuity increased) (Table 42).Mean DT and RT for pyridine, nitrobenzene and thiophene and mean DT foramyl acetate returned to or below normal levels after treatment (Table42). Only mean RT for amyl acetate did not return to normal although itwas significantly below values obtained before this treatment. Mean MEfor all odorants also returned to normal levels.

Sensory Distortions:

Mean phantageusia and phantosmia intensity decreased significantly(Table 43). In each man phantosmia disappeared.

Response Summary:

No changes in taste or smell acuity or in sensory distortion intensityoccurred in two patients immediately after treatment stimulation [(onewith head injury, one with PIHH, both women, data included in Tables 42and 43)]. These two patients were labeled non-responders. Reports of nochange in sensory distortion intensity and no change in repeat acuitytesting occurred in these two patients 2-7 d after treatment. No changeswere reported 4 wk after rTCMS I and no further data about thesepatients were obtained.

Taste and smell acuity returned to normal levels for all tastants andodorants and all sensory distortions completely disappeared in twopatients within one hr after rTCMS I [(one with head injury (one woman)one with PIHH (one man), data included in Tables 42 and 43)]. These twopatients were labeled responders. Repeat testing 2-7 d after rTCMS Idemonstrated normal sensory acuity and no sensory distortions werereported (data not shown). Reports of normal sensory acuity and absenceof any sensory distortions were received for as long as these patientswere followed (46 mo).

In the remaining 13 patients sensory acuity improved (Table 42) andsensory distortions diminished (Table 43) one hour after stimulation(Table 43). These 13 patients were also labeled responders. Symptomimprovement occurred (vs) in all responders when the field was appliedat only one skull location. Seven patients reported improvement afterleft temporoparietal stimulation, four (27%) after right temporoparietalstimulation, three (20%) after frontal stimulation and one (7%) afteroccipital stimulation. There was no improvement in the non-responders nomatter where the field was applied.

Later Post rTCMS I

Four wk-2 mo after rTCMS I, repeat testing of taste and smell acuity andmeasurement of sensory distortion intensity indicated that cognitiveimpairments had returned in 13 of the 15 responders (vs). A second trialof rTCMS (rTCMS II) was instituted in these patients.

Pre rTCMS II

Immediately prior to rTCMS II, the entire battery of sensory tests andmeasurement of sensory distortion intensity previously measured wererepeated (Table 44).

Taste:

Compared to immediately post rTCMS I, mean DTs and RTs for all tastants(except DT for HCl which did not change) increased (i.e., acuitydecreased) and mean MEs for all tastants decreased (i.e., acuitydecreased) (cf Tables 44, 42).

Smell:

Compared to immediately post rTCMS I, mean DTs and RTs for all odorants(except RT for thiophene which was lower than post rTCMS I) increased(i.e., acuity decreased) and mean MEs for all odorants decreased (i.e.,acuity decreased), (except for thiophene which was higher (cf Tables 44,42). However, mean MEs were all higher than pre rTCMS I indicating thatsome improvement after rTCMS I was retained.

Sensory Distortions:

Compared to immediately after rTCMS I, mean estimates of phantageusiaintensity increased but were significantly less than intensitiesmeasured prior to rTCMS I (cf Tables 45, 43, pre rTCMS I, 82±7, laterpost rTCMS I, 39±20, p<0.05 t test, p<0.02 paired t test). Similarlymean estimates of phantageusia intensity also increased but were lessthan pre rTCMS I (cf Tables 45, 43, pre rTCMS I, 72±14, later post rTCMSI, 47±8, p>0.05 t test, p<0.05 paired t test). Similar changes alsooccurred in phantosmia (cf Tables 45, 43).

Response Summary:

These results suggest that the improvement in cognitive impairmentswhich occurred immediately after rTCMS I persisted to some extent butthese patients “escaped” from this improvement with a return ofcognitive impairments. A second course of rTCMS was instituted.

rTCMS II

Placebo or Sham Stimulation (0.2-0.4 T):

No subjective or objective changes in either taste and/or smell acuityor in character or intensity of sensory distortions occurred in anypatient following stimulation of shoulders, neck or in any skulllocation.

Treatment Stimulation (0.8-1.1T):

Taste:

Mean DTs and RTs for all tastants decreased (i.e., acuity increased) andmean MEs for all tastants increased (i.e., acuity increased) (Table 44)as after rTCMS I (Table 42). There were significant decreases in DTs forNaCl and urea and RTs for HCl and urea. Mean MEs for all tastants werenot significantly different from normal (Table 44). DT for NaCl wassignificantly lower, (i.e., relative increased acuity) than normal aswas DT for urea.

Smell:

Mean DTs and RTs for all odorants decreased (i.e., acuity increased) andmean MEs for all odorants increased (i.e., acuity increased) (Table 44)as after rTCMS I (Table 42). There were no significant differences inmean DTs, RTs or MEs for any odorant with respect to normal. DTs fornitrobenzene and thiophene and RTs for pyridine and nitrobenzene werelower than normal (i.e., relative increased acuity) and MEs forpyridine, nitrobenzene and thiophene were higher than normal (i.e.,relative increased acuity).

Sensory Distortions:

Significant decreases occurred in phantageusia and phantosmia (Table 45)as after rTCMS I (Table 43). Phantosmia completely disappeared aftertreatment in all patients including all men and women, not just in menas after rTCMS I (Table 43). Phantageusia disappeared in 10 patients,improved by 50% in one and only slightly, if at all, in two. After rTCMSII both mean phantageusia and phantosmia intensity decreased to levelsbelow those measured later post rTCMS I (cf, Tables 43, 45).

In these 13 patients rTCMS II was effective in initiating improvementagain only at the same locus at which initial improvement occurred afterrTCMS I.

Response Summary:

After rTCMS II improvement lasted longer than after rTCMS I, lastingwk-mo. In seven of these 13 (54%) (one with head injury, four with PIHH,two idiopathic) return of sensory acuity to normal and total cessationof sensory distortions persisted for as long as measurements were made(30 mo). In the remaining six patients (one with head injury, one withPIHH, the two with idiopathic causes, the two with drug reactions)symptoms of cognitive impairment returned after one-five mo but acuitywas less impaired and sensory distortions were less intense than priorto rTCMS II (as in pre rTCMS I and II).

These six patients who “escaped” from rTCMS II were again treated withrTCMS (rTCMS III) as in rTCMS I and II. This therapy was again effectivein improving cognitive impairments but again only at the same locus thatinitiated improvement after prior stimulation (data not shown). Allsensory acuity returned to normal levels and all sensory distortionsdisappeared in these six patients for as long as they were followed(6-36 mos).

The results indicate that rTCMS was efficacious in improving tasteand/or smell acuity and in inhibiting phantosmia and phantageusia inmost patients who exhibited these symptoms. Improvement occurred afterrTCMS at one brain region. However, 13 of the 15 who initially respondedexhibited a recurrence of their symptomatology. With recurrentsymptomatology repeat rTCMS was effective again in improving symptomsbut only after application at the same site at which initial improvementoccurred, mainly the left temporoparietal region, a locus contralateralto patient handedness. This result indicates enhanced cognitiveprocessing following rTCMS in one brain locus, mainly left prefrontalcortex. Repeated stimulation in patients at this effective locusprolonged improvement in cognitive function. TCMS can be applied in manydifferent paradigms including use of single or repeated pulses, short orprolonged applications, varied wave forms, application intensity and amultiplicity of other parameters. The results of this experimentindicate that rTCMS improved sensory acuity and decreased sensorydistortions in patients with these cognitive impairments.

TABLE 42 Changes in taste and smell acuity in 17 patients withhypogeusia, hyposmia, phantosmia and/or phantageusia pre and post rTCMSI compared to normal responses TASTANT NaCl SUCROSE HCl UREA DT RT ME DTRT ME DT RT ME DT RT ME PRE 5.7 ± 0.7^(†a)   6.3 ± 0.9^(b) 26 ± 18^(d‡)4.9 ± 0.6^(c) 5.2 ± 0.6^(a) 29 ± 12^(c) 5.3 ± 0.9 6.8 ± 0.8^(a) 27 ±6^(a) 6.8 ± 1.4^(e) 9.0 ± 1.0^(a) 30 ± 9^(b) POST 3.2 ± 0.0^(g)  3.6 ±0.3^(b)* 35 ± 22 3.6 ± 0.4 3.9 ± 0.3 46 ± 12 4.1 ± 0.7 4.3 ± 0.6^(j) 57± 7^(k) 5.0 ± 1.0 5.7 ± 0.6^(h) 53 ± 8 NORMALS 3.3 ± 0.3 3.4 ± 0.2 68 ±4 3.3 ± 0.2 3.4 ± 0.2 60 ± 4 3.4 ± 0.4 3.5 ± 0.4 66 ± 4 3.6 ± 0.4 3.7 ±0.4 68 ± 4 ODORANT PYRIDINE NITROBENZENE THIOPHENE AMYL ACETATE DT RT MEDT RT ME DT RT ME DT RT ME PRE 4.0 ± 0.9^(†) 8.5 ± 0.7^(c) 35 ± 13^(e‡)6.4 ± 0.7^(b) 9.4 ± 0.4^(a) 21 ± 7^(b) 3.8 ± 0.8 7.4 ± 1.0^(a) 30 ±7^(b) 4.3 ± 1.1 8.9 ± 1.8^(a) 24 ± 7^(a) POST 1.9 ± 0.5^(ah) 4.4 ±0.9^(f) 67 ± 11 3.2 ± 0.8^(g) 6.2 ± 1.0^(g) 42 ± 8 1.9 ± 0.3^(i) 5.1 ±0.9 45 ± 7^(h) 1.4 ± 0.0^(ch) 5.2 ± 0.9^(g) 44 ± 6^(f) NORMALS 3.7 ± 0.36.0 ± 0.7 66 ± 5  3.6 ± 0.4  6.0 ± 0.6  52 ± 6 3.2 ± 0.6 3.3 ± 0.5 69 ±6  3.1 ± 0.5 3.3 ± 0.6  53 ± 5 DT, detection threshold (in BU), RT,recognition threshold (in BU), ME, magnitude estimation (in %) ^(†)MEAN± SEM (in BU) ^(‡)MEAN ± SEM (in %) *All significance determined byStudent’s t test Normals are 150 normal volunteer Compared to normals^(a)p < 0.001 ^(b)p < 0.005 ^(b)*p < 0.01 ^(c)p < 0.02 ^(d)p < 0.025^(e)p < 0.05 Compared to pre rTCMS I ^(f)p < 0.001 ^(g)p < 0.005 ^(h)p <0.01 ^(i)p < 0.025 ^(j)p < 0.02 ^(k)p < 0.05

TABLE 43 Changes in phantageusia and phantosmia in 17 patients withhyposmia, hypogeusia, phantosmia and/or phantageusia pre and post rTCMSI PHANTAGEUSIA PHANTOSMIA PATIENTS PRE POST PRE POST TOTAL(17) 82 ±7^(† ) 21 ± 7^(a) 72 ± 14   22 ± 12^(c) MEN(5) 71 ± 15   20 ± 15^(d) 70± 20 0^(b) WOMEN(12) 85 ± 6  22 ± 9^(a) 74 ± 18 33 ± 17 ( ) PatientNumber ^(†)MEAN ± SEM [in %, of most intense distortions experiencedthroughout waking state] $ Significance determined by Student's t test *All results compared to pre rTCMS I ^(a)p < 0.001 * ^(c)p < 0.02 * ^(d)p< 0.025 * ^(e)p < 0.05 * ^(b)p < 0.01

TABLE 44 Changes in taste and smell acuity in 13 patients with hyposmia,hypogeusia, phantosmia, and/or phantageusia pre and post second rTCMStreatment TASTANT NaCl(4) SUCROSE(5) HCl(8) UREA (12) DT RT ME DT RT MEDT RT ME DT RT ME PRE 5.3 ± 1.5^(†)* 9.5 ± 2.1^(a) 50 ± 10^(‡) 4.8 ±0.7^(d) 7.3 ± 1.6^(c)* 46 ± 8 4.9 ± 0.5^(c)* 8.8 ± 0.8^(a) 46 ± 8^(c)6.4 ± 0.3^(a) 7.9 ± 0.3^(a) 42 ± 10^(c)* POST 1.0 ± 0^(ae) 6.2 ± 2.8 68± 9 3.2 ± 0.7 4.7 ± 1.0 62 ± 8 4.1 ± 0.7 5.9 ± 0.9^(ch) 72 ± 6^(g) 3.4 ±0.5^(e) 4.2 ± 0.9^(hm) 63 ± 11 NORMALS 3.3 ± 0.3 3.4 ± 0.2 68 ± 4 3.3 ±0.2 3.4 ± 0.2 60 ± 4 3.4 ± 0.4 3.5 ± 0.4 66 ± 4 3.6 ± 0.4 3.7 ± 0.4 68 ±4 ODORANT PYRIDINE(9) NITROBENZENE(10) THIOPHENE(10) AMYL ACETATE(12) DTRT ME DT RT ME DT RT ME DT RT ME PRE 6.2 ± 1.2^(i†) 10.0 ± 1.1^(b) 48 ±10^(‡) 8.0 ± 1.9^(c) 10.3 ± 0.6^(e) 39 ± 8 9.0 ± 1.8^(a) 10.0 ± 0.4^(a)57 ± 9 8.7 ± 0.4^(a) 10.7 ± 0.4^(a) 36 ± 10 POST 4.8 ± 1.1  5.5 ±1.6^(i) 76 ± 5^(g) 4.2 ± 1.6  4.0 ± 1.4^(b) 62 ± 8 3.0 ± 0.9^(e)  5.0 ±1.4^(bm) 70 ± 6 4.0 ± 2.0^(I)  4.6 ± 1.5^(e) 51 ± 10 NORMALS 3.7 ± 0.3 6.0 ± 0.7 66 ± 5 3.6 ± 0.4  6.0 ± 0.6 52 ± 6 3.2 ± 0.6  3.3 ± 0.5 69 ±6 3.1 ± 0.5  3.3 ± 0.6 53 ± 5 ^(†)MEAN ± SEM (in BU) ^(‡)MEAN ± SEM (in%) *All significance determined by Student's t test Compared to normals^(a)p < 0.001 ^(b)p < 0.005 ^(c)p < 0.025 ^(l)p < 0.02 = c* ^(d)p < 0.05^(e)p < 0.001 Compared to pre rTCMS II ^(y)p < 0.01 ¢ = m* ^(f)p < 0.05^(g)p < 0.02 ^(h)p < 0.025 ^(I)p < 0.05 m: <0.005 m

TABLE 45 Changes in phantageusia and phantosmia in 13 patients withhyposmia, hypogeusia, phantosmia and/or phantageusia pre and post rTCMSII PHANTAGEUSIA PHANTOSMIA PATIENTS PRE POST PRE POST TOTAL(13)   39 ±10^(†l) 9 ± 5^(g) 47 ± 8 0^(a) MEN(4) 52 ± 15 6 ± 5^(h)  49 ± 10 0^(f)WOMEN(9) 34 ± 6^(k) 12 ± 6^(g)  44 ± 5 0^(f) ^(†)MEAN ± SEM [in %, ofmost intense distortions experienced throughout waking state] $Significance determined by Student's t test * All results compared topre rTCMS II and pre and post rTCMS I ^(a)p < 0.001 with respect to prerTCMS II and post rTCMS I ^(f)p < 0.001 compared to pre rTCMS II andpost rTCMS I ^(k)p < 0.001 compared to pre rTCMS I ^(l)p < 0.005compared to pre rTCMS I ^(g)p < 0.02 compared to pre rTCMS II ^(h)p <0.025 compared to pre rTCMS II

TABLE 46 Comparison of Biological Substances in Various Bodily FluidsNASAL MUCUS PLASMA SALIVA URINE SUBSTANCE (pg/ml) (pg/ml) (pg/ml)(pg/ml) Agout Related Protein 4.0 ± 0.9 (pg/ml) 38 ± 3 (pg/ml) 6.4 ± 1.1(pg/ml) 147 ± 20 (pg/ml) Alpha Fetoprotein (AFP) 0 (ng/ml) 2.7 ± 0.5(ng/ml) 0 (ng/ml) 0 (ng/ml) Brain Derived 8 ± 8 (ng/ml) 3676 ± 529(ng/ml) 0 (ng/ml) 0 (ng/ml) Neurotrophic Factor (BDNF) Bone Morphogenic0 (pg/ml) 4.2 (pg/ml) 0 (pg/ml) 0 (pg/ml) Protein (BMP) CiliaryNeurotrophic 0 (pg/ml) 0 (pg/ml) 0 (pg/ml) 0 (pg/ml) Factor (CNF) ESelectin 0.58 ± 0.49 (ng/ml) 20.0 ± 4.5 (ng/ml) 0 (ng/ml) 0 (ng/ml)Endoglin 0.02 ± 0.06 (ng/ml) 27 ± 0.1 (ng/ml) 0 (ng/ml) 0 (ng/ml)Endostatin 6.0 ± 1.3 (ng/ml) 102 ± 9 (ng/ml) 1.3 ± 0.3 (ng/ml) 0.4 ±0.02 (ng/ml) Endothelial Nitric Oxide 0 (ng/ml) 0 (ng/ml) 0 (ng/ml) 0(ng/ml) (ENO) Epidermal Growth Factor 7730 ± 103 (pg/ml) 52 ± 7 (pg/ml)1332 ± 258 (pg/ml) 81157 ± 11254 (pg/ml) (EGF) Erythroporetin 7.0 ± 3.7(pg/ml) 13.2 ± 2.0 (pg/ml) 0 (pg/ml) 0 (pg/ml) FAS Ligand 4.05 ± 1.9(pg/ml) 63.7 ± 8.1 (pg/ml) 0 (pg/ml) 0.73 ± 1.6 (pg/ml) FibroblasticGrowth 2.81 ± 1.3 (pg/ml) 0 (pg/ml) 0 (pg/ml) 0 (pg/ml) Factor, basic(FGF basic) inducible Nitric Oxide 19.9 ± 8.0 (U/ml) 9.5 ± 3.1 (U/ml)11.9 ± 3.6 (U/ml) 15.8 ± 7.6 (U/ml) Synthase (iNOS) Insulin-like GrowthFactor 4.2 ± 0.6 (pg/ml) — 1.3 ± 0.3 (pg/ml) — (IGF-1) IntercellularAdhesion 34 ± 4 (ng/ml) 126 ± 8 (ng/ml) 19 ± 1 (ng/ml) 20 ± 2 (ng/ml)Molecule 1 (ICAM-1) Interferon α (IFN-α 4.0 ± 1.7 (pg/ml) 2.2 ± 1.4(pg/ml) 12.7 ± 7.6 (pg/ml) 70.5 ± 16.9 (pg/ml) Interferon β (IFN β) 421± 184 (pg/ml) 866 ± 312 (pg/ml) 28 ± 14 (pg/ml) 0 (pg/ml) Interferon α(IFN γ) 85 ± 18 (pg/ml) 0.3 ± 0.3 (pg/ml) 0 (pg/ml) 0 (pg/ml) Interferonω (IFN ω) 44 ± 11 (pg/ml) 38 ± 9 (pg/ml) 0 (pg/ml) 33 ± 15 (pg/ml)Interleukin 1 (IL-1) 194 ± 24 (pg/ml) 0.1 ± 0.1 (pg/ml) 2.8 ± 1.1(pg/ml) 0.3 ± 0.2 (pg/ml) Interleukin 1 Receptor 1675 ± 517 (pg/ml)18357 ± 1922 (pg/ml) 19.7 ± 4.3 (pg/ml) 436 ± 68 (pg/ml) (IL-1R)Interleukin 2 (IL-2) 6.9 ± 5.4 (pg/ml) 0 (pg/ml) 0 (pg/ml) 0 (pg/ml)Interleukin 2 Receptor 68.8 ± 24.2 (pg/ml) 994.7 ± 101.3 (pg/ml) 11.8 ±4.6 (pg/ml) 1177.7 ± 104.1 (pg/ml) (IL-2 receptor) Interleukin 3 (IL-3)70.8 ± 23.4 (pg/ml) — 105 ± 25 (pg/ml) — Interleukin 6 (IL-6) 21.60 ±12.80 (pg/ml) 1.25 ± 0.18 (pg/ml) 0.79 ± 0.11 (pg/ml) 1.52 ± 0.43(pg/ml) Interleukin 15 (IL-15) 21.6 ± 8.9 (pg/ml) 0.67 ± 0.12 (pg/ml)0.56 ± 0.21 (pg/ml) 0.59 ± 0.36 (pg/ml) Interleukin 17 (IL-17) 2.06 ±1.18 (pg/ml) 0 (pg/ml) 1.80 ± 0.7 (pg/ml) 3.49 ± 1.44 (pg/ml)Interleukin 18 (IL-18) 270 ± 85 (pg/ml) 334 ± 52 (pg/ml) 106 ± 22(pg/ml) 0 (pg/ml) Keratinocyte Growth 15.2 ± 2.9 (pg/ml) 2.8 ± 2.8(pg/ml) 0 (pg/ml) 0 (pg/ml) Factor (KGF) L Selectin 25.35 ± 3.15 (ng/ml)20.1 ± 1.4 (ng/ml) 0 (ng/ml) 0 (ng/ml) Leptin 272 ± 70 (pg/ml) 8788 ±903 (pg/ml) 7.1 ± 1.0 (pg/ml) 0 (pg/ml) Leukemia Inhibitory 5.2 ± 3.8(pg/ml) 0 (pg/ml) 0 (pg/ml) 0 (pg/ml) Factor (LIF) Macrophage Inhibitory135 ± 27 (pg/ml) 2.8 ± 0.2 (pg/ml) 0 (pg/ml) 0 (pg/ml) Factor (MIF)Matrix Metalloproteinase 1 0.3 ± 0.1 (ng/ml) 0.8 ± 0.2 (ng/ml) 0 (ng/ml)0 (ng/ml) (MMP-1) P Selectin 1.64 ± 0.34 (pg/ml) 30.4 ± 1.7 (pg/ml) 0(pg/ml) 0 (pg/ml) Placental Growth Factor 170 ± 50 (pg/ml) 11 ± 4(pg/ml) 9 ± 3 (pg/ml) 12 ± 4 (pg/ml) (PGF) Platelet Derived Growth 420 ±63 (pg/ml) 560 ± 166 (pg/ml) 673 ± 188 (pg/ml) 0 (pg/ml) Factor (PDGF)Platelet Derived Growth 420 ± 65 (pg/ml) 560 ± 166 (pg/ml) 673 ± 188(pg/ml) 0 (pg/ml) Factor-AA (PDGF) Receptor for Advanced 0 (pg/ml) 935 ±89 (pg/ml) 0 (pg/ml) 0 (pg/ml) Glycotion End Product (RAGE) Stem CellFactor (SCF) 33.3 ± 4.6 (pg/ml) 846 ± 45 (pg/ml) — 46.4 ± 6.2 (pg/ml)Substance P 270 ± 74 (pg/ml) 250 ± 31 (pg/ml) 137 ± 47 (pg/ml) 205 ± 27(pg/ml) Thymus and Activation 1303 ± 830 (pg/ml) 875 ± 240 (pg/ml) 0(pg/ml) 0 (pg/ml) Regulated Chemokine (TARC) Tissue Inhibitor of 4326 ±553 (ng/ml) 437 ± 114 (ng/ml) 215 ± 62 (ng/ml) 0 (ng/ml)Metalloproteinase 1 (TIMP-1) TRAIL 4681 ± 464 (pg/ml) 69 ± 17 (pg/ml)685 ± 15 (pg/ml) 69 ± 12 (pg/ml) Triggering Receptor 899.9 ± 203.0(ng/ml) 83.42 ± 12.2 (ng/ml) 0.0 ± 0 (ng/ml) 0.0 ± 0 (ng/ml) Expressedon Myeloid Cells (TREM-1) Tumor Growth Factor 46 ± 8 (pg/ml) 0 (pg/ml) 0(pg/ml) 0 (pg/ml) alpha (TFG-alpha) Tumor Growth Factor β 783 ± 155(pg/ml) 339 ± 7 (pg/ml)0 363 ± 56 (pg/ml) 0 (pg/ml) (TGF β) TumorNecrosis Factor 2.2 ± 0.1 (pg/ml) 3.4 ± 0.3 (pg/ml) 0.39 ± 2.03 (pg/ml)2.0 ± 0.1 (pg/ml) alpha (TNF-alpha) Tumor Necrosis Factor 2.2 ± 0.1(pg/ml) 3.4 ± 0.3 (pg/ml) 0.4 ± 0.03 (pg/ml) 2.0 ± 0.14 (pg/ml) alphaReceptor (TNF- alpha R) Tumor Necrosis Factor β 0 (pg/ml) 0 (pg/ml) 0(pg/ml) 0 (pg/ml) (TNF β) Tumor Necrosis Factor - 938 ± 88 (pg/ml) 1583± 73 (pg/ml) 121 ± 6 (pg/ml) 1445 ± 127 (pg/ml) Receptor 1 (TNF R1)Tumor Necrosis Factor - 794 ± 78 (pg/ml) 1585 ± 73 (pg/ml) 85 ± 7(pg/ml) 1445 ± 127 (pg/ml) Receptor 2 (TNF R2) Vascular Cell Adhesion 81± 34 (ng/ml) 746 ± 91 (ng/ml) 76 ± 15 (ng/ml) 0 (ng/ml) Molecule 1(VCAM-1) Vascular Endothelial 5267 ± 714 (pg/ml) 67 ± 17 (pg/ml) 3589 ±630 (pg/ml) 69 ± 12 (pg/ml) Growth Factor (VEGF) Vascular Endothelial3449 ± 536 (pg/ml) 336.8 ± 52.1 (pg/ml) 379.8 ± 57.7 (pg/ml) — GrowthFactor C (VEGF-C) Vascular Endothelial 29.2 ± 8.2 (pg/ml) 469.0 ± 57.4(pg/ml) 6.5 ± 5.5 (pg/ml) 11.7 ± 7.9 (pg/ml) Growth Factor D (VEGF-D)Vascular Endothelial 1532 ± 445 (pg/ml) 378 ± 37 (pg/ml) 492 ± 63(pg/ml) 317 ± 39 (pg/ml) Growth Factor - Receptor 1 (VEGF-R1) VascularEndothelial 350 ± 149 (pg/ml) 5074 ± 258 (pg/ml) 99 ± 25 (pg/ml) 223 ±73 (pg/ml) Growth Factor - Receptor 2 (VEGF-R2)

TABLE 47 Examples Where Nasal Mucus Monitoring Can Replace Other AssayMethods for Predicting Future Development of Disease Prev- Currentalence Time Before Biomarker and Diagnostic of Symptoms Disease ChangeMethod Disease Appear Wilson's Tissue Copper Ceruroplasmin 95-100 1-10Years  Disease Increases level Copper level in Blood, Urine or LiverBiopsy Chronic C Reactive Blood 20  1-8 Years Obstructive ProteinPulmonary Increases Disease (COPD) Preeclampsia Endoglin Blood 3-5  2-10Weeks Increases Coronary N-Terminal-Por- Blood 5-10 Weeks-Years HeartB-Type Natri- Disease uretic Peptide (Mortality) Increases RepeatD-Dimer Present Blood 15 Weeks Thrombo- After Anti- embolism coagulationTherapy Terminated

TABLE 48 Blood Endoglin Levels in Patients that Develop PreeclampsiaCompared to Controls PREGNANCY PERIOD PREECLAMPSIA CONTROLS ONSET 46.4(ng/ml) 9.8 (ng/ml) 17-20 WEEKS 10.2 (ng/ml) 5.8 (ng/ml) 25-28 WEEKS 8.6 (ng/ml) 5.9 (ng/ml) TERM 31.0 (ng/ml) 13.3 (ng/ml) 

TABLE 49 Blood & Nasal Mucus Levels of Endoglin and Placental LikeGrowth Factor BLOOD NASAL MUCUS SUBSTANCE CONCENTRATION CONCENTRATIONENDOGLIN 12.7 ± 0.2 0.8 ± 0.5 (ng/ml) (1.1-6.0) (0.02-3.0) PLACENTAL 21± 4 372 ± 17  LIKE GROWTH  (0-79)   (32-1402) FACTOR(PlGF) (pg/ml)

TABLE 50 Comparison of Copper Levels in Patients with Taste and SmellDeficits and Controls Nasal Mucus Saliva Plasma Urine (mg/l) (mg/l)(mg/dl) (mg/24 hrs) Control 99 ± 8 22 ± 1 102 ± 10 110 ± 25 Wilson's  2± 1  3 ± 1 12 ± 2  5 ± 1 Disease Non-Wilson's 134 ± 8  28 ± 8 105 ± 20120 ± 30 Disease Patients

TABLE 51 Comparison of Zince Levels in Patients with Taste and SmellDeficits and Controls Nasal Mucus Saliva Plasma Urine (mg/l) (mg/l)(mg/dl) (mg/24 hrs) Controls  96 ± 10 89 ± 10 84 ± 12 425 ± 25Non-Wilson's Disease 236 ± 37 70 ± 20 64 ± 8  395 ± 35 Patients

What is claimed is:
 1. A diagnostic kit comprising: (a) a samplecollection device capable of obtaining a nasal specimen from a patient,wherein the nasal specimen comprises a nasal secretion, nasal mucus, ora combination thereof; (b) a diagnostic device comprising a bindingagent for detecting a biological substance or element in said nasalspecimen, wherein said biological substance or element is associatedwith a disease or condition, and wherein said disease is not arespiratory disease; and (c) written instructions for use, wherein saidbiological substance comprises agouti related protein, alpha fetoprotein(AFP), brain derived neurotrophic factor (BDNF), bone morphogeneticprotein-2 (BMP-2), ciliary neurotrophic factor (CNTF), a cyclicnucleotide, thymus and activation-regulated chemokines (CCL17/TARC) CCchemokines, cystatin, D-dimer, E selectin, endoglin, epidermal growthfactor (EGF), endothelial nitric oxide synthase (eNOS), FAS ligand,fibroblastic growth factor basic (FGF basic), granulocyte macrophagecolony stimulating factor (GM-CSF), hepatocyte growth factor (HGF),inducible nitric oxide synthase (iNOS), insulin-like growth factor 1(IGF-1), interferon alpha (INF-α), interferon beta (INF-β), interferonomega (INF-ω), intracellular adhesion molecule 1 (ICAM-1), interleukin-1receptor (IL-1 receptor), interleukin-2 receptor (IL-2 receptor),interleukin-3 (IL-3), interleukin-15 (IL-15), interleukin-17 (IL-17),interleukin-18 (IL-18), keratinocyte growth factor (KGF), L-selectin,leptin, leukemia inhibitor factor (LIF), matrix metalloproteinase 1(MVP-1), migrating inhibitory factor (MIF), nerve growth factor (NGF), Pselectin, placental growth factor (PlGF), platelet derived growthfactor-AA (PDGF-AA), platelet derived growth factor-BB (PDGF-BB), pro-Btype natiuretic peptide, receptor for abdominal glycation end product(RAGE), stem cell factor (SCF), substance P, triggering receptorexpressed on myeloid cells (TREM-1), transforming growth factor alpha(TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factorreceptor 1(TNF-R1), tumor necrosis factor receptor 2 (TNF-R2),TNF-related apoptosis-inducing ligand (TRAIL), vascular cell adhesionmolecule 1 (VCAM1), vascular endothelial growth factor C (VEGF-C),vascular growth factor D (VEGF-D), vascular endothelia growth factorreceptor 1 (VEGFR1), or vascular endothelia growth factor receptor 2(VEGFR2).
 2. The kit of claim 1, wherein said sample collection devicecomprises a swab, a wooden spatula, a bibulous material, an absorbenttip applicator, a capillary tube, or a pipette.
 3. The kit of claim 1,wherein said nasal specimen comprises nasal mucus.
 4. The kit of claim1, wherein said diagnostic device is a point of care (POC) diagnosticdevice.
 5. The kit of claim 4, wherein said POC diagnostic devicecomprises a POC nasal swab test.
 6. The kit of claim 4, wherein said POCnasal swab test comprises a colorimetric POC test performed directly onsaid swab.
 7. The kit of claim 4, wherein said POC diagnostic device isa lateral flow immunoassay.
 8. The kit of claim 1, wherein said diseaseor condition is preeclampsia, a bacterial infection, a viral infection,a parasitic infection, a metabolic disease, a gastrointestinal disease,a cardiovascular disease, a neurologic disease, a hematologic disease,an endocrine disease, a malignant disease, an autoimmune disease, achemosensory dysfunction, or an inflammatory disease.
 9. The kit ofclaim 1, wherein said biological substance is said cyclic nucleotide.10. The kit of claim 1 further comprising a phosphodiesterase (PDE)inhibitor.
 11. The kit of claim 10, wherein said PDE inhibitor istheophylline or cilostazol.
 12. The kit of claim 1, further comprising aplurality of solutions, wherein said plurality of solutions comprises astorage and transport solution, an assay solution, and a detectionsolution.
 13. The kit of claim 1, wherein said binding agent is anucleic acid, a protein, a small molecule or a combination thereof. 14.The kit of claim 9, wherein said cyclic nucleotide is cyclic GMP (cGMP).15. The kit of claim 9, wherein said cyclic nucleotide is cyclic AMP(cAMP).
 16. The kit of claim 1, wherein said disease or condition is achemosensory dysfunction that is ageusia, hypogeusia, dysgeusia,anosmia, hyposmia, or dysosmia.